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Effect of temperature, age and the pons on respiratory rhythm in the rat brainstem-spinal cord
Respiratory Physiology & Neurobiology 273 (2020) 103333
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Effect of temperature, age and the pons on respiratory rhythm in the rat brainstem-spinal cord
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M. Beth Zimmera,*, Angelina Y. Fongc, William K. Milsomb a
Department of Biological Sciences, Ferris State University, Big Rapids, MI, 49307, USA Department of Zoology, University of British Columbia, Vancouver, BC, V6R 1ZT, Canada c Department of Physiology, University of Melbourne, Parkville, Victoria, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Respiration Pons Temperature Postnatal development Rat
Neonatal animals are extremely tolerant of hypothermia. However, cooling will ultimately lead to ventilatory arrest, or cessation of respiratory movements. Upon rewarming, ventilation can recover spontaneously (autoresuscitation). This study examined the effect of age (P0-P5) and the pons on respiratory-related output during hypothermic ventilatory arrest and recovery using a brainstem-spinal cord preparation of neonatal rats. As temperature fell, burst frequency slowed, burst duration increased, burst shape became fragmented and eventually respiratory arrest occurred in all preparations. Removing the pons had little effect on younger preparations (P0-P2). Older preparations (P4-P5) with the pons removed continued to burst at cooler temperatures compared to pons-intact preparations and burst durations were significantly longer. Episodic breathing patterns were observed in all preparations (all ages, pons on or off) at lower temperatures. At 27 °C, however, episodic breathing was only observed in younger preparations with the pons on. These data suggest that developmental changes occurring at the level of the pons underlie the loss of hypothermic tolerance and episodic breathing.
1. Introduction In adult mammals, severe hypothermia causes decreases in ventilation and heart rate, which lead to ventilatory and cardiac arrest and ultimately death. The body temperature at which ventilation and heart rate decrease is age-dependent and neonates have a significantly greater tolerance to hypothermia than adults. Adolph (1948a) found that although severe unregulated reductions in temperature (2–5 °C) caused ventilatory and cardiac arrest in neonatal rat pups, rewarming resulted in the spontaneous recovery of both ventilation and heart rate with no apparent adverse effects. In adult mammals, hypothermia also leads to ventilatory and cardiac arrest, however, adults do not autoresuscitate and very little time is needed before adverse effects occur (Adolph, 1948b, 1951). The dramatic difference between the responses to hypothermia in neonates and adults has led to the speculation that the response of the neonate may be an evolutionary adaptation in which accidental cooling may occur when the mother leaves the nest or burrow; this adaptation would aid in energy conservation when the mother (i.e., the food source) is removed (Hill, 2000). Despite this intriguing physiology of neonatal mammals, few studies have examined the underlying mechanisms of hypothermic respiratory arrest or the spontaneous autoresuscitation that occurs during rewarming.
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The in vitro brainstem spinal cord preparation was first described by Suzue (1984) and was promoted as a means to investigate neural mechanisms regulating respiration including the effects of pH, temperature, and neurotransmitter activation and blockade (Murakoshi et al., 1985). Murakoshi et al. (1985) showed that the optimal temperature for this preparation was around 28 °C and that increasing or decreasing the temperature of the bath reduced respiratory frequency. Using an in vitro brainstem-spinal cord preparation, Mellen et al. (2002) found that respiratory frequency decreased during cooling and arrested around 17 °C. They found that the arrest of respiratory motor output was due to failure at the level of the respiratory rhythm generator and not due to interrupted neural transmission to the phrenic motoneurons (Mellen et al., 2002). However, intact neonates of similar ages did not show respiratory arrest until temperature fell to 10 °C or lower (Tattersall and Milsom, 2003). The difference in temperature at which respiration stopped between the intact animal and the reduced brainstem-spinal cord preparation was speculated to be due to tonic excitatory inputs present in vivo (Tattersall and Milsom, 2003). In rats, the loss of the ability to autoresuscitate is relatively quick and by three weeks of age, pups show a mature, adult response to hypothermia where breathing stops at a temperature of 17–25 °C and does not spontaneously restart (Adolph, 1951; Corcoran et al., 2012).
Corresponding author at: Department of Biological Sciences, Ferris State University, 820 Campus Drive, ASC 2004; Big Rapids, MI, 49307, USA. E-mail address: [email protected] (M.B. Zimmer).
https://doi.org/10.1016/j.resp.2019.103333 Received 22 February 2019; Received in revised form 25 September 2019; Accepted 16 October 2019 Available online 18 October 2019 1569-9048/ Published by Elsevier B.V.
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2.2. Experiment 1: cooling and warming with the pons intact or removed
The present study was designed to further our understanding of the effect of hypothermia on respiration; specifically how differences in neonatal age and pontine inputs affect respiratory rhythm and pattern during hypothermia, respiratory arrest or the stoppage of respiratory activity, and autoresuscitation. Using the brainstem-spinal cord preparation of the neonatal rat, Corcoran and Milsom (2009) found that the influence of the pons on respiratory frequency changes from excitatory at P0 to inhibitory at P4. During the same developmental period, there is a reduction in the length of the viability of the brainstem-spinal cord preparation, which varied between the medullary or the pontomedullary preparations (Fong et al., 2008). The changes in respiratory motor output during this developmental period are not due to changes in tissue mass or central hypoxia/anoxia, but rather changes in the development of respiratory neural control mechanisms (Fong et al., 2008). Even the earliest studies using the brainstem-spinal cord preparation suggested that the “gasping-like” nature of the respiratory motor output was due to the deafferentation of the preparation rather than hypoxia (Murakoshi et al., 1985). Other studies have also demonstrated that the pons provides both excitatory and inhibitory inputs to medullary respiratory nuclei to affect respiration (Hilaire et al., 1989; Errchidi et al., 1990, 1991; Zuperku et al., 2018). During fetal development, the pattern of breathing movements is not continuous. Studies suggest that the regulation of irregular and episodic patterns of breathing observed in utero is due to alternating excitatory and inhibitory inputs (Hilaire and Duron, 1999). Based on this information, we hypothesized that as animals develop we would observe a decrease in episodic respiratory patterns. We also hypothesized that as greater inhibitory pontine inputs develop, the ability of the respiratory system to autoresuscitate from hypothermia would decrease. Therefore, we predicted that the youngest preparations would have greater tolerances to the cold compared to older preparations and that respiratory arrest would occur when the inhibition arising from the pons was the greatest, i.e., when the pons remained intact in the older preparations. Removal of the pons, and thus the inhibitory inputs, should allow for the continued excitation of the respiratory system in older preparations at colder temperatures.
The brainstem-spinal cord preparation was pinned ventral side up in a recording chamber made of molded Plexiglas with a stainless steel grid separating the chamber into an upper and lower compartment. The preparation was oriented such that the superfusate flowed over the brainstem prior to the spinal cord. The temperature of the oxygenated aCSF was regulated with a Lauda cooler (Model RC6) and administered to an upper and lower chamber at a rate of 2.5 ml/min with a single outflow rate of 5 ml/min. This ensured adequate superfusion of the brainstem-spinal cord tissue and allowed for fine temperature control of the aCSF. The brainstem-spinal cord preparations were dissected at room temperature (19–25 °C) and respiratory neural activity was recorded for 20–30 minutes. All data collected from 1 to 2 day old neonates were grouped together to create a "young" neonate group (P1 = 9; P2 = 3) and data from 4 to 5 day old neonates were grouped together to create an "old" neonate group (P4 = 9; P5 = 2). In each group, roughly half of the preparations were run with the pons intact (young group n = 6; old group n = 5) and half with the pons transected at the ponto-medullary border (young group n = 6; old group n = 6). Each preparation was then warmed to a starting value of 27 °C and baseline activity recorded for another 20–30 minutes. Following this, a step-wise cooling and rewarming protocol was applied in which temperature was changed in steps of 4 °C, each steady temperature recording lasting 20–30 minutes (27 °C, 23 °C, 19 °C, 15 °C). Cooling in steps continued until respiratoryrelated activity stopped. Following 30 min with no activity, the preparation was rewarmed in the same steps. It took approximately 10 min to reach a new equilibrium with each 4 °C step (cooling or heating). 2.3. Experiment 2: chemical and mechanical removal of the pons To specifically examine the role of the pons in producing episodic breathing, pontomedullary preparations (n = 10) were set up in a splitbath arrangement as described in Fong et al. (2008). The acrylic recording dish consisted of two chambers joined by a narrow gap. The preparation was placed in the chamber with the caudal cerebellar artery aligned at the narrow gap, and Vaseline (grease) was packed in the gap to completely separate the two chambers, with one chamber containing the pons and the other containing the medulla and spinal cord. Each chamber had its own perfusion circuit (5–9 ml/min each) allowing for separate superfusion of the pons and medulla (see Fong et al., 2008; Corcoran and Milsom, 2009 for details). At the end of the stabilization period, the superfusate to the pontine chamber was replaced with one containing 0.2 mM Ca2+ and 5 mM Mg2+ (low Ca2+/high Mg2+) for 45–60 min (pH 7.4) to chemically inhibit the pontine inputs. After superfusion with low Ca2+/high Mg2+, the pontine superfusate was switched back to regular aCSF for 20 min to ensure that all drug was washed out of the pontine chamber and tubing. Following washout of the low Ca2+/high Mg2+, the Vaseline was removed and the pons was manually transected using a razor blade at the same level as the grease gap. The pontine tissue mass was not removed following transection but left in situ in the chamber; the descending inputs were severed but the total tissue mass in the perfusion chamber was not altered. The preparation was again allowed to stabilize and fictive breathing was recorded for 30 min. All experiments in this series were performed on younger preparations (P0-2) at 27 °C.
2. Methods 2.1. Animal care and tissue preparation All experiments were performed with prior approval from the University of British Columbia Animal Care Committee acting under the guidelines of the Canadian Council for Animal Care (CCAC) (UBC ACC# A01-0034). Neonatal Sprague-Dawley rats (1–5 days old) were purchased from the Animal Care Breeding Facility at the University of British Columbia. They were placed into a chamber and deeply anesthetized with 2–4% halothane or isoflurane. The animals were quickly and crudely dissected to isolate the brainstem-spinal cord, which was immersed in artificial cerebral spinal fluid (aCSF) (composed of 113 mM sodium chloride, 3 mM potassium chloride, 1.2 mM sodium phosphate, 1.5 mM calcium chloride, 1 mM magnesium chloride, 30 mM sodium bicarbonate, and 30 mM dextrose and equilibrated with 95% oxygen/5% carbon dioxide (pH 7.4)). Under a dissecting microscope, any remaining dura or tissue was removed, the pons was either left intact or removed depending on the protocol (see below), and the spinal cord was cut at the 7th cervical root. The brainstem-spinal cord preparations were placed ventral side up into a recording chamber (see below for descriptions of the two different chambers that were used). A glass suction electrode was attached to the spinal roots (C1 or C4) to measure respiratory-related motor output associated with the inspiratory phase of ventilation. A thermistor was placed in the bath next to the brainstem to monitor the bath temperature. Electrical signals from suction electrodes were amplified, filtered (100 Hz – 3 kHz) and recorded at 2000 samples / second using WindaqPro computer data acquisition software (DI200AC; DataQ Instruments, Akron, OH, USA).
2.4. Experiment 3: oxygenation of the brainstem-spinal cord during cooling and warming In order to verify that oxygenation of the medullary respiratory centers was not different between pons-intact and pons-removed preparations during cooling and rewarming, we ran an extra set of rats (pons intact n = 6; pons removed n = 8) at a single age (2 days old). In 2
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Fig. 1. Histograms showing the respiratory neural discharge of the brainstem-spinal cord preparations of different ages throughout steady state temperature changes during cooling and rewarming. The black bars represent the pons-intact preparations while the gray bars represent the pons-removed preparations. At 27 °C, the burst frequency of the older pons-removed preparations was significantly higher than that of the older, pons intact and young preparations. An (*) marks a significant effect of temperature compared to starting values of burst frequency, burst duration and burst area.
Instruments). The microsensors were calibrated at two points, watervapor saturated air and oxygen-free solution. Water-vapor saturated air was achieved by placing wet cotton pellets in a container with two access holes for the Optode microsensor and the temperature sensor. The container was allowed to equilibrate for 10 min to ensure complete saturation of air before calibration. Oxygen-free solution was made by bubbling 10 ml of aCSF with 100% nitrogen for ∼10 min. in a small container with two access holes for the Optode microsensor and the
this set of experiments, respiratory-related motor output was monitored to determine when the preparation was rhythmically active, when respiratory arrest occurred, and when it spontaneously restarted. The preparation was warmed to 27 °C and allowed to stabilize for 30 min prior to PO2 measurements. Measurement of medullary PO2 was performed using a fiber-optic microsensor with a tip diameter of ∼30 μm that allowed for high spatial resolution (Optode, OxyMicro System, World Precision 3
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(2) the last 5 min of low Ca2+/high Mg2+, (3) the last five minutes of the recovery period, and (4) 30 min after pontine transection. The average PO2 measurements at each degree Celsius were taken and analyzed using two-way repeated measures ANOVA to compare the effect of cooling in the presence and absence of the pons. All statistics were performed using Systat software (version 10.2) and all data are presented as means ± SEM. 3. Results 3.1. Experiment 1: the effect of cooling and warming with the pons intact or removed Fig. 2. Graph showing the relationship between the respiratory frequency and the bath temperature at the start of the experiment (27 °C) versus the bath temperature at which the last respiratory burst occurred. The older, pons removed preparation had a significantly greater frequency at 27 °C than the younger preparation (#) and the older, pons intact preparation (*). The temperature at which the respiratory frequency stopped was significantly lower in the older, pons-removed preparations compared to all of the other groups (δ).
Both age (P = 0.022) and the pons (P = 0.003) significantly influenced the baseline frequency of the respiratory motor discharge of brainstem-spinal cord preparations recorded at a bath temperature of 27°C. The pons had no effect on the frequency of the motor output in young preparations. However, in preparations from older neonates (P45), the respiratory burst frequency was significantly greater in the pons removed compared to pons-intact preparations. The fictive breathing frequency of the older preparations with the pons removed was also significantly greater compared to the young preparations with the pons removed. Despite the observed differences in burst frequency at 27°C, there was no significant effect of age or the pons on the burst duration of the motor output (Fig. 1). Cooling the brainstem-spinal cord preparation resulted in a progressive, significant decrease in respiratory motor activity (Fig. 1) and ended in respiratory arrest in all preparations. The temperature at which the last respiratory motor activity was recorded was significantly influenced by age and the pons (P = 0.0002) (Fig. 2). In young preparations, the temperature at which respiratory activity ceased was similar in pons off and pons intact preparations, however, in older preparations, the temperature at which respiratory activity ceased was lower in the pons off preparations (P = 0.001). The higher the starting frequency, the lower the temperature at which respiratory arrest occurred. Upon rewarming, respiratory activity spontaneously returned and the frequency returned to control values at 27°C in all preparations (Fig. 1). At 27 °C, there was no significant effect of age or the pons on burst duration, however during cooling, there was a significant effect on burst duration. Decreasing temperature resulted in a significant increase in burst duration in both age groups with the pons-removed (P = 0.006). After respiratory arrest, the burst duration in the pons intact preparations was significantly greater during rewarming, but was not significantly different from the pons-removed preparations (Fig. 1). With rewarming back to 27 °C, burst duration decreased back to control values in all preparations. Similar changes were observed in burst area (P = 0.008) although only the burst area of young pons removed preparations were elevated during cooling, while during rewarming, the burst area was elevated in both young and old preparations with the pons removed. Burst area returned to starting values after they were rewarmed back to 27°C. While a similar trend was observed for changes in burst amplitude, the changes were not significant (Fig. 1). Before respiratory arrest and during the initial stages of warming, the shape of the individual bursts of motor discharge became disrupted and irregular in appearance, or fragmented (Fig. 3). This was observed in all preparations at all ages regardless of the shape of the respiratory motor burst output (decrementing or bell-shaped motor output) or whether the pons was present or not. At 19 °C, the respiratory activity of the old, pons intact preparations had ceased and the number of bursts recorded in the young pons on and removed preparations were very low and fragmented (Fig. 3). Thus, this data was not considered reliable for determining an accurate burst duration and amplitude. However, the data did show the same general trend in burst duration and amplitude at 19 °C although with greater variability (data not shown). The respiratory motor output of brainstem-spinal cord preparations
temperature sensor. PO2 measurements were taken at the approximate level of the preBotC in both pons intact and pons removed preparations. The optode microsensor was mounted in a stereotaxic frame in an electrode holder attached to a hydraulic microdrive to enable fine control in the vertical plane. The tip of the microsensor was placed rostrocaudally at the level corresponding to the preBotC; the tip was aligned with the most rostral hypoglossal nerve rootlet and moved +1.0 mm lateral to midline and a depth of 400μM, (Smith et al., 1990; Ruangkittisakul et al., 2007). Cooling and rewarming of the brainstem-spinal cord preparation occurred while simultaneously recording PO2 and respiratory-motor output. The time for cooling and rewarming was approximately 1 °/ 2 min. PO2 measurements were taken every 5 s and were acquired directly by data acquisition software. All PO2 measurements were temperature corrected. 2.5. Data analysis To analyze the effect of temperature on respiratory motor output, all raw nerve recordings were full-wave rectified and integrated (time constant = 100 ms) using Windaq Playback Software and the Advanced Codas analyses package (DataQ Instruments, Akron, OH, USA). Integrated signals were analyzed for respiratory motor burst duration, burst peak amplitude, burst area and total burst frequency. At least 30 individual bursts were measured for each temperature except when the frequency became extremely slow and the complete 20–30 minutes was analyzed. Burst amplitude and burst area of nerve recordings were normalized to the data collected at 27 °C. The effect of hypothermia on fictive respiratory frequency was analyzed using a multivariate repeated measures analysis of variance (ANOVA) in which the pons and age were considered the main effects and temperature was the dependent variable. At reduced temperatures, preparations that stopped producing a motor output were assigned a zero frequency in the calculation of the mean frequency. To determine the effect of hypothermia on burst amplitude, area and duration, only bursts that were present could be examined, and as a result the sample size decreased with reducing temperatures. Therefore, we only examined the effect of hypothermia on burst amplitude, area and duration at two temperatures, 27 °C and 23 °C, during cooling and rewarming. Finally, the respiratory pattern was also characterized for each trace at each temperature. Patterns were classified as regular where bursts were evenly spaced throughout the trace or episodic in which two or more bursts were clustered together. For the split bath preparations, nerve recordings were analyzed as above over 5 min periods at four different time points: (1) at the end of the 30 min stabilization period (baseline), 4
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Fig. 3. Representative raw (top) and integrated (bottom) respiratory-related nerve activity from the first cervical (C1) ventral roots of neonatal brainstem-spinal cord preparations showing the effect of cooling on the burst profile. We observed consistent fragmentation of the motor burst at the coldest bath temperatures (Tb) before respiratory arrest and during rewarming. This was observed in both pons intact and pons removed preparations of young and old neonates. The expanded traces on the right side are denoted with an asterisk (*).
(Fig. 6). While removal of the pons led to an increase in PO2 at lower temperatures, this difference was not significant. Values returned to baseline during rewarming of the brainstem-spinal cord preparation. There was a slight hysteresis observed during rewarming due to a small transient difference between the bath temperature and the temperature of the brain.
exhibited either a regular pattern in which respiratory-related burst of neural activity were evenly spaced from one burst to the next, or they exhibited an episodic pattern in which bursts of neural activity were clustered together into two (most common) or more bursts per episode (Fig. 4). Immediately after the brainstem-spinal cord was prepared and recording commenced at room temperature (19–25 °C), episodic patterns were observed in all preparations of all ages, with or without the pons. Many of the young brainstem-spinal cord preparations (pons on and off combined) exhibited episodic respiratory activity initially at room temperature (11 out of 12). When the preparations were warmed to 27 °C, the episodic pattern converted to a regular pattern in most preparations. The two preparations that continued to produce episodes had the pons intact. During the cooling protocol, many of the preparations become episodic again (7 out of 12). In the older preparations, only 3 out of 11 preparations exhibited the episodic pattern at room temperature immediately after preparing the preparation. As the tissue was warmed to 27 °C, the episodic pattern converted to a regular pattern in all of the older preparations (100%). When the older preparations were cooled, one of the preparations exhibited episodes again.
4. Discussion It was previously shown that the respiratory network of the isolated neonatal rat brainstem-spinal cord preparation undergoes respiratory arrest during hypothermia and is capable of spontaneous recovery upon rewarming (Mellen et al., 2002). The present study shows that the cold tolerance of this preparation and the age at which respiratory arrest occurs varies with the age of the neonate and inputs arising from the pons. This study further demonstrates the role of the pons in the production of episodic breathing in younger rat preparations. Episodic breathing patterns are exhibited by all mammals during development and are well described in the literature (Milsom, 1991; Milsom et al., 1997). While most adult mammals breathe in a continuous pattern, some mammals continue to breathe in episodes throughout adulthood, e.g., whales, seals (in water and on land) and many mammals in hibernation. The underlying neural mechanisms controlling and modulating the episodic breathing patterns are not well understood. Studies of breathing patterns during hibernation suggest that temperature, the neural state (awake, sleep, hibernation) and inputs from the pons may all play a role in producing episodic breathing (Milsom et al., 2004; Milsom and Jackson, 2011). This study shows that reduction in temperature and inputs from the pons are also important in the production of episodic breathing in neonatal rats.
3.2. Experiment 2: Chemical and mechanical removal of the pons The importance of the pons for the production of episodic breathing in the younger preparations at 27 °C was explored further using the split bath preparation and only preparations that were exhibiting episodes were studied. Initially, 80% of the bursts occurred within an episode and about 20% of the bursts appeared independently or not closely associated with a burst. When the pons-intact preparation was bathed with the low Ca2+/high Mg2+ solution, the incidence of episodes was greatly reduced such that the majority of the bursts occurred in singles and only the very rare episode of 2 bursts were visible. When the pons was subsequently transected, the episodes were eliminated altogether (Fig. 5).
4.1. Effect of hypothermia and rewarming on fictive respiratory frequency At 27 °C (our baseline condition), descending inputs from the pons significantly reduced the fictive respiratory frequency of the older brainstem-spinal cord preparations but not the younger preparations. This is consistent with results from other studies (Corcoran and Milsom, 2009; Fong et al., 2008; Hilaire et al., 1989, 2004; Errchidi et al., 1990, 1991). Corcoran and Milsom (2009) reported that at P0 the pons provides an excitatory input that disappears by P2 and is replaced by an
3.3. Experiment 3: the effect of temperature on oxygenation of the brain tissue (preBötC) Progressive cooling of the brainstem-spinal cord of P2 neonates resulted in a significant increase in PO2 at the level of the preBötC 5
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Fig. 4. Representative integrated (top) and raw (bottom) respiratory-related nerve activity from the first cervical (C1) ventral roots of neonatal brainstem-spinal cord preparations showing the episodic (A, B, C) versus continuous (D, E, F) respiratory patterns that were observed at room temperature. Panels B and E show expanded versions of the areas highlighted in grey in panels A and D, while panels C and F are Poincaré plots of the interburst intervals from 3 different preparations (as indicated by the different symbols) showing the clustering of bursts in the episodic pattern and a single cluster in the continuous pattern.
frequency than younger preparations at 27 °C (Zimmer and Milsom, 2004). This suggests that the older medullary preparations may simply have a greater cold tolerance, independent of the starting frequency and tonic activity. Interestingly, hamsters retain the ability to autoresuscitate as adults (Corcoran et al., 2012; Gajda et al., 2010). It is not clear if this is related to the cold tolerance of neurons in different species. The different effects of cold on respiratory frequency observed in our young versus old in vitro preparations between P0-P5 are not observed in vivo, however. Tattersall and Milsom (2003) found that in neonate rat pups of similar ages (P1-P5; in vivo, non-anesthetized), the progressive fall in burst frequency (and increase in tidal volume) was similar regardless of age. The only age difference that they observed was in the time it took for body temperature to fall during cooling.
inhibitory input by P4. Other studies have also observed early excitatory effects of the pons on respiratory frequency in embryos (Borday et al., 1997) and in fetuses (Di Pasquale et al., 1994; Hilaire and Duron, 1999) that are replaced by a net inhibitory input to the medullary respiratory centers during development (Hilaire et al., 1989, 2004; Errchidi et al., 1990, 1991). During cooling, respiratory frequency decreased in all preparations, young and old, with and without the pons intact, just as it does in vivo (Adolph, 1948a; Fairchild, 1948; Tattersall and Milsom, 2003). The data from this study suggest that the temperature at which respiratory arrest occurs is related to the burst frequency of the preparation under baseline conditions, i.e., the higher the starting frequency, and presumably the greater the level of tonic drive, the lower the temperature at which respiratory arrest occurs. Del Negro et al. (2005) showed that changes in the ionic currents that underlie respiratory rhythmogenesis during development enhance neuronal excitability and produce greater tonic drive within the respiratory rhythmogenic centers. A previous study on hamster brainstem-spinal cord preparations, however, found that although older preparations with the pons removed also continued to discharge at lower temperatures before respiratory arrest occurred, the older hamster preparations had a significantly lower starting
4.2. The effect of age and the pons on episodic pattern during cooling and rewarming When we prepared the brainstem-spinal cords and started recording at room temperature, we discovered that a high percentage of young brainstem-spinal cord preparations and a lower percentage of old 6
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Fig. 5. Representative raw (bottom) and integrated (top) respiratory-related nerve activity from the first cervical (C1) ventral roots of a split-bath preparation of a neonatal brainstem-spinal cord showing an episodic breathing pattern under baseline (27 °C) conditions (top left) followed by chemical inhibition with a low Ca2+/high Mg2+ solution (top right), return to regular aCSF (bottom left) and manual transection of the pons.
again went from episodic to a regular, continuous pattern. Our observations suggest that developmental age, temperature and inputs from the pons all influence the expression of the episodic pattern. The mechanisms that underlie the production of episodic breathing remain unknown. On the one hand, it has been shown that opioids will induce episodic breathing via quantal slowing resulting from transmission failure of rhythmic drive from pre-I neurons to pre-BotC I networks (Mellen et al., 2003). On the other hand, it has recently been shown that inhibition of pre-I neurons in the parafacial respiratory group (pFRG) by galanin converts episodic breathing to a pattern of regularly spaced bursts (Bautista et al., 2018). Clearly pre-I neurons, which receive input from pontine respiratory neurons, are implicated in the control of breathing episodes, but the nature of the interactions requires further research.
preparations exhibited episodic patterns of respiratory motor output. When the preparations were warmed to 27 °C, the pattern changed to a regular, continuous breathing pattern in all of the preparations except most of the younger preparations with the pons intact. During cooling, the episodic pattern returned in many of the preparations. In brainstemspinal cord preparations from hamsters, fictive breathing was only observed in preparations with the pons removed (no respiratory activity was recorded with the pons intact). However, respiratory motor bursts became episodic during rapid, continuous cooling in a large percentage of young preparations (70%), while in older preparations, episodic patterns of activity were never observed (Zimmer and Milsom, 2004). The hamster preparations did not exhibit episodic breathing during slower, step-wise cooling and rewarming to the same temperatures (Zimmer and Milsom, 2004). Mellen et al. (2002) also observed episodic patterns of motor discharge using neonatal rat preparations that employed rapid, progressive cooling, but only during the rewarming phase of experiments. At this point, the relative roles of species, age and the nature of the cooling-rewarming protocol on the production of breathing episodes is unclear. We further examined the role of the pons in P2 rat brainstem-spinal cord preparations. We found that at 27 °C, episodic breathing was normal for pons intact preparations. When we chemically blocked the inputs with a low Ca2+/high Mg2+ solution, the pattern changed to regular, continuous pattern and the pattern could be reversed with wash-out of the solution. When we transected the pons, the pattern
4.3. The effect of age and the pons on burst duration during cooling and rewarming It is widely recognized that inputs from pontine nuclei and the vagus are involved in inspiratory timing (Dick et al., 2008; Cohen and Shaw, 2004). We found that at 27 °C removal of the pons did not affect burst duration in young or old preparations, however, the presence of the pons did affect burst duration during cooling and rewarming. During cooling (23 °C), burst duration increased in all of the pons-removed preparations, but not when the pons remained intact. This Fig. 6. Graphs showing the PO2 in the region of the pre-Bötzinger complex during cooling (white symbols, black symbols denote no difference from 27 °C) and rewarming (gray symbols) of brainstem spinal cord preparations with and without the pons intact (P2, younger neonates). As the temperature decreased there was a significant increase in the PO2 near the region where respiratory neurons are located.
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suggests that there may be inhibitory inputs arising from the pons that, during cooling, limit the length of the inspiratory burst. During the autoresuscitation following respiratory arrest, the burst duration increased in all preparations, both with and without the pons-intact. Consistent with our findings, Tattersall and Milsom (2003) found that the first breaths during rewarming of neonatal rats in vivo were of very long duration, but were not gasps. Tattersall and Milsom (2003) speculated that after respiratory arrest there may be small airway collapse and that a large tidal volume may be necessary to re-inflate the lungs. This may indeed be true, but the data from the present study further suggest that the long duration breaths that are observed during autoresuscitation may arise directly from rewarming the respiratory neural circuitry. This may reflect reactivation of the rhythm-generating sites in the medulla before other modulatory respiratory inputs, specifically those arising from pontine nuclei. This is consistent with the proposal by Bartlett (1955) suggesting that the pons is inhibited first by cold, and may return later than respiratory centers in the medulla during rewarming.
occurred during rewarming and respiratory activity returned to baseline levels. Episodic breathing patterns were observed in all preparations, more so in younger preparations (P1-P2). Episodic breathing was eliminated by warming in older preparations, but also required elimination of the pons in conjunction with warming in the younger preparations. Older preparations were more cold tolerant when the pons was removed. It thus appears that maturational changes in the pons in rat pups underlie the loss of both cold tolerance and episodic breathing. Our data also indicate that the effects of postnatal age or pontine removal on respiratory pattern in these experiments were not due to changes in oxygen availability to the medullary respiratory network.
4.4. The effect of temperature on oxygenation of the brain tissue (preBötC)
Adolph, E.F., 1948a. Tolerance to cold and anoxia in infant rats. Am. J. Physiol. 155, 366–377. Adolph, E.F., 1948b. Lethal limits of cold tolerance in adult rats. Am. J. Physiol. 155, 378–387. Adolph, E.F., 1951. Responses to hypothermia in several species of infant mammal. Am. J. Physiol. 166, 75–91. Bartlett Jr., R.G., 1955. Apneustic breathing produced by severe hypothermia with mild anesthesia. Am. J. Physiol. 180 (2), 433–437. Bautista, T.G., Fong, A.Y., Pilowsky, P.M., Ikeda, K., Kawakami, K., Spirovski, D., Onimaru, H., 2018. The Expression of galanin in the parafacial respiratory group and its effects on respiration in neonatal rats. Neuroscience 384, 1–13. Benedetti, G.M., Silverstein, F.S., 2018. Targeted temperature management in pediatric care. Pediatr. Neurol. 88, 12–24. Borday, V., Fortin, G., Champagnat, J., 1997. Early ontogeny of rhythm generation and control of breathing. Respir. Physiol. 110, 245–249. Cohen, M.I., Shaw, C.F., 2004. Role in the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons. Respir. Physiol. Neurobiol. 143 (2-3), 127–140. Corcoran, A.E., Milsom, W.K., 2009. Maturational changes in pontine and medullary alpha-adrenoceptor influences on respiratory rhythm generation in neonatal rats. Respir. Physiol. Neurobiol. 165, 195–201. Corcoran, A.E., Andrade, D.V., Marshall, L.H., Milsom, W.K., 2012. Developmental changes in cold tolerance and ability to autoresuscitate from hypothermic respiratory arrest are not linked in rats and hamsters. Respir. Physiol. Neurobiol. 181, 249–258. Del Negro, C.A., Morgado-Valle, C., Hayes, J.A., Mackay, D.D., Pace, R.W., Crowder, E.A., Feldman, J.L., 2005. Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation. J. Neurosci. 25 (2), 446–453. Di Pasquale, E., Monteau, R., Hilaire, G., 1994. Endogenous serotonin modulates the fetal respiratory rhythm: an in vitro study in the rat. Brain Res. Dev. Brain Res. 80, 222–232. Dick, T.E., Shannon, R., Lindsey, B.G., Nuding, S.C., Segers, L.S., Baekey, D.M., Morris, K.F., 2008. Pontine respiratory-modulated activity before and after vagotomy in decerebrate cats. J. Physiol. 586 (17), 4265–4282. Errchidi, S., Hilaire, G., Monteau, R., 1990. Permanent release of noradrenalin modulates respiratory frequency in the newborn rat: an in vitro study. J. Physiol. (Lond.) 429, 497–510. Errichidi, S., Monteau, R., Hilaire, G., 1991. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study. J. Physiol. (Lond.) 443, 477–498. Fairchild, J., 1948. Effects of cold on infant rats: body temperatures, oxygen consumption, electrocardiograms. Am. J. Physiol. 155, 355–365. Fong, A.Y., Corcoran, A.E., Zimmer, M.B., Andrade, D.V., Milsom, W.K., 2008. Respiratory rhythm of brainstem-spinal cord preparations: effects of maturation, age, mass and oxygenation. Respir. Physiol. Neurobiol. 164, 429–440. Gajda, B.M., Fong, A.Y., Milsom, W.K., 2010. Species differences in respiratory rhythm generation in rodents. Am. J. Physiol. 298, 887–898. Hilaire, G., Duron, B., 1999. Maturation of the mammalian respiratory system. Physiol. Rev. 79 (2), 325–332. Hilaire, G., Monteau, R., Errchidi, S., 1989. Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat. Brain Res. 485, 325–332. Hilaire, G., Viemari, J.-C., Coulon, P., Simonneau, M., Bevengut, M., 2004. Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir. Physiol. Neurobiol. 143, 187–197. Hill, R.W., 2000. Anoxia tolerance to oxygen necessity: paradigm shift in the physiology of survival of apneic deep hypothermia in neonatal rodents. In: Heldmaier, G., Klingenspor, M. (Eds.), Life in the Cold, Eleventh International Hibernation Symposium. Springer, Heidelberg, Germany. pp. 199–205. Kieboom, J.K., Verkade, H.J., Burgerhof, J.G., Bierens, J.J., van Rheenen, P.F., Kneyber, M.C., Albers, M.J., 2015. Outcome after resuscitation beyond 30 minutes in drowned children with cardiac arrest and hypothermia: dutch nationwide retrospective cohort
Acknowledgement This work was funded by the NSERC of Canada (W.K. Milsom, 587150). References
Our study supports other studies that demonstrate that the brainstem-spinal cord preparation is not hypoxic with or without the pons (Fong et al., 2008; Murakoshi et al., 1985). Murakoshi et al. (1985) demonstrated that the change from a bell-shaped burst to a gasp-like decrementing burst pattern was due to the removal of sensory afferents and argued that it was not due to hypoxia. Fong et al., 2008 showed that the surface of the brainstem-spinal cord is hyperoxic and at the level of the preBötzinger complex, O2 levels were around 100 Torr. They also showed no differences in oxygen at three different positions relative to midline. The present study showed that the levels of oxygen were ∼40 Torr at the level of the preBötzinger complex at 27 °C, however during cooling the levels of O2 increased with the decreasing temperature. Our results show that there is an excess of oxygen in the brainstem at colder temperatures; further evidence that hypoxia and subsequent gasping are not involved in reinitiating the respiratory cycle during rewarming. This is not surprising since oxygen solubility increases as temperature decreases. Tissue oxygen levels could also be higher due to a decrease in cellular metabolism in the cold. Despite the increased oxygen supply in the brainstem regions, we observed a consistent fragmentation of the burst shape at the lowest temperatures during our stepwise cooling and rewarming. This was also observed by Mellen et al. (2002) but only during the transient rewarming phase in their experiments. Since they observed this irregular respiratory output from both field potentials around the preBötzinger complex and the phrenic motor output, they speculated that the fragmentation or irregular discharge originated from a lack of synchronization within the network in the medulla and not from disruption of the descending motor drive. 4.5. Conclusions Targeted temperature management for the treatment of acute brain injury is being widely used to enhance survival of neonates and children in clinical settings (Benedetti and Silverstein, 2018). Case studies show varying degrees of clinical outcomes after accidental hypothermia and drowning. A Dutch retrospective study showed that maintaining resuscitation efforts past 30 min results in poor outcomes in drowned victims with hypothermia and cardiac arrest (Kieboom et al. (2015). Understanding temperature effects on neural function and the specific developmental changes in respiratory function that occur during the early neonatal period are critical to help develop better therapeutic hypothermia strategies and to help determine when resuscitation efforts may be useful. Cooling neonatal rat brainstem–spinal cord preparations reduced respiratory frequency followed by respiratory arrest. Autoresuscitation 8
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Ruangkittisakul, A., Secchia, L., Bornes, T.D., Palathinkal, D.M., Ballanyi, K., 2007. Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem. J. Physiol. 584 (2), 489–508. Smith, J.C., Greer, J.J., Liu, G.S., Feldman, J.L., 1990. Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 64 (4) 1149-1146. Suzue, T., 1984. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol. 354, 173–183. Tattersall, G., Milsom, W.K., 2003. Hypothermia-induced respiratory arrest and recovery in neonatal rats. Respir. Physiol. Neurobiol. 137 (1), 29–40. Zimmer, M.B., Milsom, W.K., 2004. Effect of hypothermia on respiratory rhythm generation in hamster brainstem-spinal cord preparations. Respir. Physiol. Neurobiol. 142, 237–249. Zuperku, E.J., Stucke, A.G., Krolikowski, J.G., Tomlinson, J., Hopp, F.A., Stuth, E.A., 2018. Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency. Respir. Physiol. Neurobiol. https://doi.org/10.1016/j.resp. 2018.06.011.
study. The BMJ (Clinical Research ed.) 350, h418. https://doi.org/10.1136/bmj. h418. Mellen, N.M., Janczewski, W.A., Bocchiaro, C.M., Feldman, J.L., 2003. Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation. Neuron 37, 821–826. Mellen, N.M., Milsom, W.K., Feldman, J.L., 2002. Hypothermia and recovery from respiratory arrest in a neonatal rat in vitro brainstem preparation. Am. J. Physiol. 282, R484–491. Milsom, W.K., 1991. Intermittent breathing. Annu. Rev. Physiol. 53, 87–105. Milsom, W.K., Chatburn, J., Zimmer, M.B., 2004. Pontine influences on respiratory control in ectothermic and heterothermic vertebrates. Resp. Physiol. Neurobiol. 143, 263–280. Milsom, W.K., Harris, M.B., Reid, S.G., 1997. Do descending influences alternate to produce episodic breathing? Respir. Physiol. 110, 307–317. Milsom, W.K., Jackson, D., 2011. Hibernation and gas exchange. Compr. Physiol. 2011 (1), 397–420. Murakoshi, T., Suzue, T., Tamai, S., 1985. A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat. Br. J. Pharmac. 86, 95–104.
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Effect of temperature, age and the pons on respiratory rhythm in the rat brainstem-spinal cord
T
M. Beth Zimmera,*, Angelina Y. Fongc, William K. Milsomb a
Department of Biological Sciences, Ferris State University, Big Rapids, MI, 49307, USA Department of Zoology, University of British Columbia, Vancouver, BC, V6R 1ZT, Canada c Department of Physiology, University of Melbourne, Parkville, Victoria, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Respiration Pons Temperature Postnatal development Rat
Neonatal animals are extremely tolerant of hypothermia. However, cooling will ultimately lead to ventilatory arrest, or cessation of respiratory movements. Upon rewarming, ventilation can recover spontaneously (autoresuscitation). This study examined the effect of age (P0-P5) and the pons on respiratory-related output during hypothermic ventilatory arrest and recovery using a brainstem-spinal cord preparation of neonatal rats. As temperature fell, burst frequency slowed, burst duration increased, burst shape became fragmented and eventually respiratory arrest occurred in all preparations. Removing the pons had little effect on younger preparations (P0-P2). Older preparations (P4-P5) with the pons removed continued to burst at cooler temperatures compared to pons-intact preparations and burst durations were significantly longer. Episodic breathing patterns were observed in all preparations (all ages, pons on or off) at lower temperatures. At 27 °C, however, episodic breathing was only observed in younger preparations with the pons on. These data suggest that developmental changes occurring at the level of the pons underlie the loss of hypothermic tolerance and episodic breathing.
1. Introduction In adult mammals, severe hypothermia causes decreases in ventilation and heart rate, which lead to ventilatory and cardiac arrest and ultimately death. The body temperature at which ventilation and heart rate decrease is age-dependent and neonates have a significantly greater tolerance to hypothermia than adults. Adolph (1948a) found that although severe unregulated reductions in temperature (2–5 °C) caused ventilatory and cardiac arrest in neonatal rat pups, rewarming resulted in the spontaneous recovery of both ventilation and heart rate with no apparent adverse effects. In adult mammals, hypothermia also leads to ventilatory and cardiac arrest, however, adults do not autoresuscitate and very little time is needed before adverse effects occur (Adolph, 1948b, 1951). The dramatic difference between the responses to hypothermia in neonates and adults has led to the speculation that the response of the neonate may be an evolutionary adaptation in which accidental cooling may occur when the mother leaves the nest or burrow; this adaptation would aid in energy conservation when the mother (i.e., the food source) is removed (Hill, 2000). Despite this intriguing physiology of neonatal mammals, few studies have examined the underlying mechanisms of hypothermic respiratory arrest or the spontaneous autoresuscitation that occurs during rewarming.
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The in vitro brainstem spinal cord preparation was first described by Suzue (1984) and was promoted as a means to investigate neural mechanisms regulating respiration including the effects of pH, temperature, and neurotransmitter activation and blockade (Murakoshi et al., 1985). Murakoshi et al. (1985) showed that the optimal temperature for this preparation was around 28 °C and that increasing or decreasing the temperature of the bath reduced respiratory frequency. Using an in vitro brainstem-spinal cord preparation, Mellen et al. (2002) found that respiratory frequency decreased during cooling and arrested around 17 °C. They found that the arrest of respiratory motor output was due to failure at the level of the respiratory rhythm generator and not due to interrupted neural transmission to the phrenic motoneurons (Mellen et al., 2002). However, intact neonates of similar ages did not show respiratory arrest until temperature fell to 10 °C or lower (Tattersall and Milsom, 2003). The difference in temperature at which respiration stopped between the intact animal and the reduced brainstem-spinal cord preparation was speculated to be due to tonic excitatory inputs present in vivo (Tattersall and Milsom, 2003). In rats, the loss of the ability to autoresuscitate is relatively quick and by three weeks of age, pups show a mature, adult response to hypothermia where breathing stops at a temperature of 17–25 °C and does not spontaneously restart (Adolph, 1951; Corcoran et al., 2012).
Corresponding author at: Department of Biological Sciences, Ferris State University, 820 Campus Drive, ASC 2004; Big Rapids, MI, 49307, USA. E-mail address: [email protected] (M.B. Zimmer).
https://doi.org/10.1016/j.resp.2019.103333 Received 22 February 2019; Received in revised form 25 September 2019; Accepted 16 October 2019 Available online 18 October 2019 1569-9048/ Published by Elsevier B.V.
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2.2. Experiment 1: cooling and warming with the pons intact or removed
The present study was designed to further our understanding of the effect of hypothermia on respiration; specifically how differences in neonatal age and pontine inputs affect respiratory rhythm and pattern during hypothermia, respiratory arrest or the stoppage of respiratory activity, and autoresuscitation. Using the brainstem-spinal cord preparation of the neonatal rat, Corcoran and Milsom (2009) found that the influence of the pons on respiratory frequency changes from excitatory at P0 to inhibitory at P4. During the same developmental period, there is a reduction in the length of the viability of the brainstem-spinal cord preparation, which varied between the medullary or the pontomedullary preparations (Fong et al., 2008). The changes in respiratory motor output during this developmental period are not due to changes in tissue mass or central hypoxia/anoxia, but rather changes in the development of respiratory neural control mechanisms (Fong et al., 2008). Even the earliest studies using the brainstem-spinal cord preparation suggested that the “gasping-like” nature of the respiratory motor output was due to the deafferentation of the preparation rather than hypoxia (Murakoshi et al., 1985). Other studies have also demonstrated that the pons provides both excitatory and inhibitory inputs to medullary respiratory nuclei to affect respiration (Hilaire et al., 1989; Errchidi et al., 1990, 1991; Zuperku et al., 2018). During fetal development, the pattern of breathing movements is not continuous. Studies suggest that the regulation of irregular and episodic patterns of breathing observed in utero is due to alternating excitatory and inhibitory inputs (Hilaire and Duron, 1999). Based on this information, we hypothesized that as animals develop we would observe a decrease in episodic respiratory patterns. We also hypothesized that as greater inhibitory pontine inputs develop, the ability of the respiratory system to autoresuscitate from hypothermia would decrease. Therefore, we predicted that the youngest preparations would have greater tolerances to the cold compared to older preparations and that respiratory arrest would occur when the inhibition arising from the pons was the greatest, i.e., when the pons remained intact in the older preparations. Removal of the pons, and thus the inhibitory inputs, should allow for the continued excitation of the respiratory system in older preparations at colder temperatures.
The brainstem-spinal cord preparation was pinned ventral side up in a recording chamber made of molded Plexiglas with a stainless steel grid separating the chamber into an upper and lower compartment. The preparation was oriented such that the superfusate flowed over the brainstem prior to the spinal cord. The temperature of the oxygenated aCSF was regulated with a Lauda cooler (Model RC6) and administered to an upper and lower chamber at a rate of 2.5 ml/min with a single outflow rate of 5 ml/min. This ensured adequate superfusion of the brainstem-spinal cord tissue and allowed for fine temperature control of the aCSF. The brainstem-spinal cord preparations were dissected at room temperature (19–25 °C) and respiratory neural activity was recorded for 20–30 minutes. All data collected from 1 to 2 day old neonates were grouped together to create a "young" neonate group (P1 = 9; P2 = 3) and data from 4 to 5 day old neonates were grouped together to create an "old" neonate group (P4 = 9; P5 = 2). In each group, roughly half of the preparations were run with the pons intact (young group n = 6; old group n = 5) and half with the pons transected at the ponto-medullary border (young group n = 6; old group n = 6). Each preparation was then warmed to a starting value of 27 °C and baseline activity recorded for another 20–30 minutes. Following this, a step-wise cooling and rewarming protocol was applied in which temperature was changed in steps of 4 °C, each steady temperature recording lasting 20–30 minutes (27 °C, 23 °C, 19 °C, 15 °C). Cooling in steps continued until respiratoryrelated activity stopped. Following 30 min with no activity, the preparation was rewarmed in the same steps. It took approximately 10 min to reach a new equilibrium with each 4 °C step (cooling or heating). 2.3. Experiment 2: chemical and mechanical removal of the pons To specifically examine the role of the pons in producing episodic breathing, pontomedullary preparations (n = 10) were set up in a splitbath arrangement as described in Fong et al. (2008). The acrylic recording dish consisted of two chambers joined by a narrow gap. The preparation was placed in the chamber with the caudal cerebellar artery aligned at the narrow gap, and Vaseline (grease) was packed in the gap to completely separate the two chambers, with one chamber containing the pons and the other containing the medulla and spinal cord. Each chamber had its own perfusion circuit (5–9 ml/min each) allowing for separate superfusion of the pons and medulla (see Fong et al., 2008; Corcoran and Milsom, 2009 for details). At the end of the stabilization period, the superfusate to the pontine chamber was replaced with one containing 0.2 mM Ca2+ and 5 mM Mg2+ (low Ca2+/high Mg2+) for 45–60 min (pH 7.4) to chemically inhibit the pontine inputs. After superfusion with low Ca2+/high Mg2+, the pontine superfusate was switched back to regular aCSF for 20 min to ensure that all drug was washed out of the pontine chamber and tubing. Following washout of the low Ca2+/high Mg2+, the Vaseline was removed and the pons was manually transected using a razor blade at the same level as the grease gap. The pontine tissue mass was not removed following transection but left in situ in the chamber; the descending inputs were severed but the total tissue mass in the perfusion chamber was not altered. The preparation was again allowed to stabilize and fictive breathing was recorded for 30 min. All experiments in this series were performed on younger preparations (P0-2) at 27 °C.
2. Methods 2.1. Animal care and tissue preparation All experiments were performed with prior approval from the University of British Columbia Animal Care Committee acting under the guidelines of the Canadian Council for Animal Care (CCAC) (UBC ACC# A01-0034). Neonatal Sprague-Dawley rats (1–5 days old) were purchased from the Animal Care Breeding Facility at the University of British Columbia. They were placed into a chamber and deeply anesthetized with 2–4% halothane or isoflurane. The animals were quickly and crudely dissected to isolate the brainstem-spinal cord, which was immersed in artificial cerebral spinal fluid (aCSF) (composed of 113 mM sodium chloride, 3 mM potassium chloride, 1.2 mM sodium phosphate, 1.5 mM calcium chloride, 1 mM magnesium chloride, 30 mM sodium bicarbonate, and 30 mM dextrose and equilibrated with 95% oxygen/5% carbon dioxide (pH 7.4)). Under a dissecting microscope, any remaining dura or tissue was removed, the pons was either left intact or removed depending on the protocol (see below), and the spinal cord was cut at the 7th cervical root. The brainstem-spinal cord preparations were placed ventral side up into a recording chamber (see below for descriptions of the two different chambers that were used). A glass suction electrode was attached to the spinal roots (C1 or C4) to measure respiratory-related motor output associated with the inspiratory phase of ventilation. A thermistor was placed in the bath next to the brainstem to monitor the bath temperature. Electrical signals from suction electrodes were amplified, filtered (100 Hz – 3 kHz) and recorded at 2000 samples / second using WindaqPro computer data acquisition software (DI200AC; DataQ Instruments, Akron, OH, USA).
2.4. Experiment 3: oxygenation of the brainstem-spinal cord during cooling and warming In order to verify that oxygenation of the medullary respiratory centers was not different between pons-intact and pons-removed preparations during cooling and rewarming, we ran an extra set of rats (pons intact n = 6; pons removed n = 8) at a single age (2 days old). In 2
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Fig. 1. Histograms showing the respiratory neural discharge of the brainstem-spinal cord preparations of different ages throughout steady state temperature changes during cooling and rewarming. The black bars represent the pons-intact preparations while the gray bars represent the pons-removed preparations. At 27 °C, the burst frequency of the older pons-removed preparations was significantly higher than that of the older, pons intact and young preparations. An (*) marks a significant effect of temperature compared to starting values of burst frequency, burst duration and burst area.
Instruments). The microsensors were calibrated at two points, watervapor saturated air and oxygen-free solution. Water-vapor saturated air was achieved by placing wet cotton pellets in a container with two access holes for the Optode microsensor and the temperature sensor. The container was allowed to equilibrate for 10 min to ensure complete saturation of air before calibration. Oxygen-free solution was made by bubbling 10 ml of aCSF with 100% nitrogen for ∼10 min. in a small container with two access holes for the Optode microsensor and the
this set of experiments, respiratory-related motor output was monitored to determine when the preparation was rhythmically active, when respiratory arrest occurred, and when it spontaneously restarted. The preparation was warmed to 27 °C and allowed to stabilize for 30 min prior to PO2 measurements. Measurement of medullary PO2 was performed using a fiber-optic microsensor with a tip diameter of ∼30 μm that allowed for high spatial resolution (Optode, OxyMicro System, World Precision 3
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(2) the last 5 min of low Ca2+/high Mg2+, (3) the last five minutes of the recovery period, and (4) 30 min after pontine transection. The average PO2 measurements at each degree Celsius were taken and analyzed using two-way repeated measures ANOVA to compare the effect of cooling in the presence and absence of the pons. All statistics were performed using Systat software (version 10.2) and all data are presented as means ± SEM. 3. Results 3.1. Experiment 1: the effect of cooling and warming with the pons intact or removed Fig. 2. Graph showing the relationship between the respiratory frequency and the bath temperature at the start of the experiment (27 °C) versus the bath temperature at which the last respiratory burst occurred. The older, pons removed preparation had a significantly greater frequency at 27 °C than the younger preparation (#) and the older, pons intact preparation (*). The temperature at which the respiratory frequency stopped was significantly lower in the older, pons-removed preparations compared to all of the other groups (δ).
Both age (P = 0.022) and the pons (P = 0.003) significantly influenced the baseline frequency of the respiratory motor discharge of brainstem-spinal cord preparations recorded at a bath temperature of 27°C. The pons had no effect on the frequency of the motor output in young preparations. However, in preparations from older neonates (P45), the respiratory burst frequency was significantly greater in the pons removed compared to pons-intact preparations. The fictive breathing frequency of the older preparations with the pons removed was also significantly greater compared to the young preparations with the pons removed. Despite the observed differences in burst frequency at 27°C, there was no significant effect of age or the pons on the burst duration of the motor output (Fig. 1). Cooling the brainstem-spinal cord preparation resulted in a progressive, significant decrease in respiratory motor activity (Fig. 1) and ended in respiratory arrest in all preparations. The temperature at which the last respiratory motor activity was recorded was significantly influenced by age and the pons (P = 0.0002) (Fig. 2). In young preparations, the temperature at which respiratory activity ceased was similar in pons off and pons intact preparations, however, in older preparations, the temperature at which respiratory activity ceased was lower in the pons off preparations (P = 0.001). The higher the starting frequency, the lower the temperature at which respiratory arrest occurred. Upon rewarming, respiratory activity spontaneously returned and the frequency returned to control values at 27°C in all preparations (Fig. 1). At 27 °C, there was no significant effect of age or the pons on burst duration, however during cooling, there was a significant effect on burst duration. Decreasing temperature resulted in a significant increase in burst duration in both age groups with the pons-removed (P = 0.006). After respiratory arrest, the burst duration in the pons intact preparations was significantly greater during rewarming, but was not significantly different from the pons-removed preparations (Fig. 1). With rewarming back to 27 °C, burst duration decreased back to control values in all preparations. Similar changes were observed in burst area (P = 0.008) although only the burst area of young pons removed preparations were elevated during cooling, while during rewarming, the burst area was elevated in both young and old preparations with the pons removed. Burst area returned to starting values after they were rewarmed back to 27°C. While a similar trend was observed for changes in burst amplitude, the changes were not significant (Fig. 1). Before respiratory arrest and during the initial stages of warming, the shape of the individual bursts of motor discharge became disrupted and irregular in appearance, or fragmented (Fig. 3). This was observed in all preparations at all ages regardless of the shape of the respiratory motor burst output (decrementing or bell-shaped motor output) or whether the pons was present or not. At 19 °C, the respiratory activity of the old, pons intact preparations had ceased and the number of bursts recorded in the young pons on and removed preparations were very low and fragmented (Fig. 3). Thus, this data was not considered reliable for determining an accurate burst duration and amplitude. However, the data did show the same general trend in burst duration and amplitude at 19 °C although with greater variability (data not shown). The respiratory motor output of brainstem-spinal cord preparations
temperature sensor. PO2 measurements were taken at the approximate level of the preBotC in both pons intact and pons removed preparations. The optode microsensor was mounted in a stereotaxic frame in an electrode holder attached to a hydraulic microdrive to enable fine control in the vertical plane. The tip of the microsensor was placed rostrocaudally at the level corresponding to the preBotC; the tip was aligned with the most rostral hypoglossal nerve rootlet and moved +1.0 mm lateral to midline and a depth of 400μM, (Smith et al., 1990; Ruangkittisakul et al., 2007). Cooling and rewarming of the brainstem-spinal cord preparation occurred while simultaneously recording PO2 and respiratory-motor output. The time for cooling and rewarming was approximately 1 °/ 2 min. PO2 measurements were taken every 5 s and were acquired directly by data acquisition software. All PO2 measurements were temperature corrected. 2.5. Data analysis To analyze the effect of temperature on respiratory motor output, all raw nerve recordings were full-wave rectified and integrated (time constant = 100 ms) using Windaq Playback Software and the Advanced Codas analyses package (DataQ Instruments, Akron, OH, USA). Integrated signals were analyzed for respiratory motor burst duration, burst peak amplitude, burst area and total burst frequency. At least 30 individual bursts were measured for each temperature except when the frequency became extremely slow and the complete 20–30 minutes was analyzed. Burst amplitude and burst area of nerve recordings were normalized to the data collected at 27 °C. The effect of hypothermia on fictive respiratory frequency was analyzed using a multivariate repeated measures analysis of variance (ANOVA) in which the pons and age were considered the main effects and temperature was the dependent variable. At reduced temperatures, preparations that stopped producing a motor output were assigned a zero frequency in the calculation of the mean frequency. To determine the effect of hypothermia on burst amplitude, area and duration, only bursts that were present could be examined, and as a result the sample size decreased with reducing temperatures. Therefore, we only examined the effect of hypothermia on burst amplitude, area and duration at two temperatures, 27 °C and 23 °C, during cooling and rewarming. Finally, the respiratory pattern was also characterized for each trace at each temperature. Patterns were classified as regular where bursts were evenly spaced throughout the trace or episodic in which two or more bursts were clustered together. For the split bath preparations, nerve recordings were analyzed as above over 5 min periods at four different time points: (1) at the end of the 30 min stabilization period (baseline), 4
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Fig. 3. Representative raw (top) and integrated (bottom) respiratory-related nerve activity from the first cervical (C1) ventral roots of neonatal brainstem-spinal cord preparations showing the effect of cooling on the burst profile. We observed consistent fragmentation of the motor burst at the coldest bath temperatures (Tb) before respiratory arrest and during rewarming. This was observed in both pons intact and pons removed preparations of young and old neonates. The expanded traces on the right side are denoted with an asterisk (*).
(Fig. 6). While removal of the pons led to an increase in PO2 at lower temperatures, this difference was not significant. Values returned to baseline during rewarming of the brainstem-spinal cord preparation. There was a slight hysteresis observed during rewarming due to a small transient difference between the bath temperature and the temperature of the brain.
exhibited either a regular pattern in which respiratory-related burst of neural activity were evenly spaced from one burst to the next, or they exhibited an episodic pattern in which bursts of neural activity were clustered together into two (most common) or more bursts per episode (Fig. 4). Immediately after the brainstem-spinal cord was prepared and recording commenced at room temperature (19–25 °C), episodic patterns were observed in all preparations of all ages, with or without the pons. Many of the young brainstem-spinal cord preparations (pons on and off combined) exhibited episodic respiratory activity initially at room temperature (11 out of 12). When the preparations were warmed to 27 °C, the episodic pattern converted to a regular pattern in most preparations. The two preparations that continued to produce episodes had the pons intact. During the cooling protocol, many of the preparations become episodic again (7 out of 12). In the older preparations, only 3 out of 11 preparations exhibited the episodic pattern at room temperature immediately after preparing the preparation. As the tissue was warmed to 27 °C, the episodic pattern converted to a regular pattern in all of the older preparations (100%). When the older preparations were cooled, one of the preparations exhibited episodes again.
4. Discussion It was previously shown that the respiratory network of the isolated neonatal rat brainstem-spinal cord preparation undergoes respiratory arrest during hypothermia and is capable of spontaneous recovery upon rewarming (Mellen et al., 2002). The present study shows that the cold tolerance of this preparation and the age at which respiratory arrest occurs varies with the age of the neonate and inputs arising from the pons. This study further demonstrates the role of the pons in the production of episodic breathing in younger rat preparations. Episodic breathing patterns are exhibited by all mammals during development and are well described in the literature (Milsom, 1991; Milsom et al., 1997). While most adult mammals breathe in a continuous pattern, some mammals continue to breathe in episodes throughout adulthood, e.g., whales, seals (in water and on land) and many mammals in hibernation. The underlying neural mechanisms controlling and modulating the episodic breathing patterns are not well understood. Studies of breathing patterns during hibernation suggest that temperature, the neural state (awake, sleep, hibernation) and inputs from the pons may all play a role in producing episodic breathing (Milsom et al., 2004; Milsom and Jackson, 2011). This study shows that reduction in temperature and inputs from the pons are also important in the production of episodic breathing in neonatal rats.
3.2. Experiment 2: Chemical and mechanical removal of the pons The importance of the pons for the production of episodic breathing in the younger preparations at 27 °C was explored further using the split bath preparation and only preparations that were exhibiting episodes were studied. Initially, 80% of the bursts occurred within an episode and about 20% of the bursts appeared independently or not closely associated with a burst. When the pons-intact preparation was bathed with the low Ca2+/high Mg2+ solution, the incidence of episodes was greatly reduced such that the majority of the bursts occurred in singles and only the very rare episode of 2 bursts were visible. When the pons was subsequently transected, the episodes were eliminated altogether (Fig. 5).
4.1. Effect of hypothermia and rewarming on fictive respiratory frequency At 27 °C (our baseline condition), descending inputs from the pons significantly reduced the fictive respiratory frequency of the older brainstem-spinal cord preparations but not the younger preparations. This is consistent with results from other studies (Corcoran and Milsom, 2009; Fong et al., 2008; Hilaire et al., 1989, 2004; Errchidi et al., 1990, 1991). Corcoran and Milsom (2009) reported that at P0 the pons provides an excitatory input that disappears by P2 and is replaced by an
3.3. Experiment 3: the effect of temperature on oxygenation of the brain tissue (preBötC) Progressive cooling of the brainstem-spinal cord of P2 neonates resulted in a significant increase in PO2 at the level of the preBötC 5
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Fig. 4. Representative integrated (top) and raw (bottom) respiratory-related nerve activity from the first cervical (C1) ventral roots of neonatal brainstem-spinal cord preparations showing the episodic (A, B, C) versus continuous (D, E, F) respiratory patterns that were observed at room temperature. Panels B and E show expanded versions of the areas highlighted in grey in panels A and D, while panels C and F are Poincaré plots of the interburst intervals from 3 different preparations (as indicated by the different symbols) showing the clustering of bursts in the episodic pattern and a single cluster in the continuous pattern.
frequency than younger preparations at 27 °C (Zimmer and Milsom, 2004). This suggests that the older medullary preparations may simply have a greater cold tolerance, independent of the starting frequency and tonic activity. Interestingly, hamsters retain the ability to autoresuscitate as adults (Corcoran et al., 2012; Gajda et al., 2010). It is not clear if this is related to the cold tolerance of neurons in different species. The different effects of cold on respiratory frequency observed in our young versus old in vitro preparations between P0-P5 are not observed in vivo, however. Tattersall and Milsom (2003) found that in neonate rat pups of similar ages (P1-P5; in vivo, non-anesthetized), the progressive fall in burst frequency (and increase in tidal volume) was similar regardless of age. The only age difference that they observed was in the time it took for body temperature to fall during cooling.
inhibitory input by P4. Other studies have also observed early excitatory effects of the pons on respiratory frequency in embryos (Borday et al., 1997) and in fetuses (Di Pasquale et al., 1994; Hilaire and Duron, 1999) that are replaced by a net inhibitory input to the medullary respiratory centers during development (Hilaire et al., 1989, 2004; Errchidi et al., 1990, 1991). During cooling, respiratory frequency decreased in all preparations, young and old, with and without the pons intact, just as it does in vivo (Adolph, 1948a; Fairchild, 1948; Tattersall and Milsom, 2003). The data from this study suggest that the temperature at which respiratory arrest occurs is related to the burst frequency of the preparation under baseline conditions, i.e., the higher the starting frequency, and presumably the greater the level of tonic drive, the lower the temperature at which respiratory arrest occurs. Del Negro et al. (2005) showed that changes in the ionic currents that underlie respiratory rhythmogenesis during development enhance neuronal excitability and produce greater tonic drive within the respiratory rhythmogenic centers. A previous study on hamster brainstem-spinal cord preparations, however, found that although older preparations with the pons removed also continued to discharge at lower temperatures before respiratory arrest occurred, the older hamster preparations had a significantly lower starting
4.2. The effect of age and the pons on episodic pattern during cooling and rewarming When we prepared the brainstem-spinal cords and started recording at room temperature, we discovered that a high percentage of young brainstem-spinal cord preparations and a lower percentage of old 6
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Fig. 5. Representative raw (bottom) and integrated (top) respiratory-related nerve activity from the first cervical (C1) ventral roots of a split-bath preparation of a neonatal brainstem-spinal cord showing an episodic breathing pattern under baseline (27 °C) conditions (top left) followed by chemical inhibition with a low Ca2+/high Mg2+ solution (top right), return to regular aCSF (bottom left) and manual transection of the pons.
again went from episodic to a regular, continuous pattern. Our observations suggest that developmental age, temperature and inputs from the pons all influence the expression of the episodic pattern. The mechanisms that underlie the production of episodic breathing remain unknown. On the one hand, it has been shown that opioids will induce episodic breathing via quantal slowing resulting from transmission failure of rhythmic drive from pre-I neurons to pre-BotC I networks (Mellen et al., 2003). On the other hand, it has recently been shown that inhibition of pre-I neurons in the parafacial respiratory group (pFRG) by galanin converts episodic breathing to a pattern of regularly spaced bursts (Bautista et al., 2018). Clearly pre-I neurons, which receive input from pontine respiratory neurons, are implicated in the control of breathing episodes, but the nature of the interactions requires further research.
preparations exhibited episodic patterns of respiratory motor output. When the preparations were warmed to 27 °C, the pattern changed to a regular, continuous breathing pattern in all of the preparations except most of the younger preparations with the pons intact. During cooling, the episodic pattern returned in many of the preparations. In brainstemspinal cord preparations from hamsters, fictive breathing was only observed in preparations with the pons removed (no respiratory activity was recorded with the pons intact). However, respiratory motor bursts became episodic during rapid, continuous cooling in a large percentage of young preparations (70%), while in older preparations, episodic patterns of activity were never observed (Zimmer and Milsom, 2004). The hamster preparations did not exhibit episodic breathing during slower, step-wise cooling and rewarming to the same temperatures (Zimmer and Milsom, 2004). Mellen et al. (2002) also observed episodic patterns of motor discharge using neonatal rat preparations that employed rapid, progressive cooling, but only during the rewarming phase of experiments. At this point, the relative roles of species, age and the nature of the cooling-rewarming protocol on the production of breathing episodes is unclear. We further examined the role of the pons in P2 rat brainstem-spinal cord preparations. We found that at 27 °C, episodic breathing was normal for pons intact preparations. When we chemically blocked the inputs with a low Ca2+/high Mg2+ solution, the pattern changed to regular, continuous pattern and the pattern could be reversed with wash-out of the solution. When we transected the pons, the pattern
4.3. The effect of age and the pons on burst duration during cooling and rewarming It is widely recognized that inputs from pontine nuclei and the vagus are involved in inspiratory timing (Dick et al., 2008; Cohen and Shaw, 2004). We found that at 27 °C removal of the pons did not affect burst duration in young or old preparations, however, the presence of the pons did affect burst duration during cooling and rewarming. During cooling (23 °C), burst duration increased in all of the pons-removed preparations, but not when the pons remained intact. This Fig. 6. Graphs showing the PO2 in the region of the pre-Bötzinger complex during cooling (white symbols, black symbols denote no difference from 27 °C) and rewarming (gray symbols) of brainstem spinal cord preparations with and without the pons intact (P2, younger neonates). As the temperature decreased there was a significant increase in the PO2 near the region where respiratory neurons are located.
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suggests that there may be inhibitory inputs arising from the pons that, during cooling, limit the length of the inspiratory burst. During the autoresuscitation following respiratory arrest, the burst duration increased in all preparations, both with and without the pons-intact. Consistent with our findings, Tattersall and Milsom (2003) found that the first breaths during rewarming of neonatal rats in vivo were of very long duration, but were not gasps. Tattersall and Milsom (2003) speculated that after respiratory arrest there may be small airway collapse and that a large tidal volume may be necessary to re-inflate the lungs. This may indeed be true, but the data from the present study further suggest that the long duration breaths that are observed during autoresuscitation may arise directly from rewarming the respiratory neural circuitry. This may reflect reactivation of the rhythm-generating sites in the medulla before other modulatory respiratory inputs, specifically those arising from pontine nuclei. This is consistent with the proposal by Bartlett (1955) suggesting that the pons is inhibited first by cold, and may return later than respiratory centers in the medulla during rewarming.
occurred during rewarming and respiratory activity returned to baseline levels. Episodic breathing patterns were observed in all preparations, more so in younger preparations (P1-P2). Episodic breathing was eliminated by warming in older preparations, but also required elimination of the pons in conjunction with warming in the younger preparations. Older preparations were more cold tolerant when the pons was removed. It thus appears that maturational changes in the pons in rat pups underlie the loss of both cold tolerance and episodic breathing. Our data also indicate that the effects of postnatal age or pontine removal on respiratory pattern in these experiments were not due to changes in oxygen availability to the medullary respiratory network.
4.4. The effect of temperature on oxygenation of the brain tissue (preBötC)
Adolph, E.F., 1948a. Tolerance to cold and anoxia in infant rats. Am. J. Physiol. 155, 366–377. Adolph, E.F., 1948b. Lethal limits of cold tolerance in adult rats. Am. J. Physiol. 155, 378–387. Adolph, E.F., 1951. Responses to hypothermia in several species of infant mammal. Am. J. Physiol. 166, 75–91. Bartlett Jr., R.G., 1955. Apneustic breathing produced by severe hypothermia with mild anesthesia. Am. J. Physiol. 180 (2), 433–437. Bautista, T.G., Fong, A.Y., Pilowsky, P.M., Ikeda, K., Kawakami, K., Spirovski, D., Onimaru, H., 2018. The Expression of galanin in the parafacial respiratory group and its effects on respiration in neonatal rats. Neuroscience 384, 1–13. Benedetti, G.M., Silverstein, F.S., 2018. Targeted temperature management in pediatric care. Pediatr. Neurol. 88, 12–24. Borday, V., Fortin, G., Champagnat, J., 1997. Early ontogeny of rhythm generation and control of breathing. Respir. Physiol. 110, 245–249. Cohen, M.I., Shaw, C.F., 2004. Role in the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons. Respir. Physiol. Neurobiol. 143 (2-3), 127–140. Corcoran, A.E., Milsom, W.K., 2009. Maturational changes in pontine and medullary alpha-adrenoceptor influences on respiratory rhythm generation in neonatal rats. Respir. Physiol. Neurobiol. 165, 195–201. Corcoran, A.E., Andrade, D.V., Marshall, L.H., Milsom, W.K., 2012. Developmental changes in cold tolerance and ability to autoresuscitate from hypothermic respiratory arrest are not linked in rats and hamsters. Respir. Physiol. Neurobiol. 181, 249–258. Del Negro, C.A., Morgado-Valle, C., Hayes, J.A., Mackay, D.D., Pace, R.W., Crowder, E.A., Feldman, J.L., 2005. Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation. J. Neurosci. 25 (2), 446–453. Di Pasquale, E., Monteau, R., Hilaire, G., 1994. Endogenous serotonin modulates the fetal respiratory rhythm: an in vitro study in the rat. Brain Res. Dev. Brain Res. 80, 222–232. Dick, T.E., Shannon, R., Lindsey, B.G., Nuding, S.C., Segers, L.S., Baekey, D.M., Morris, K.F., 2008. Pontine respiratory-modulated activity before and after vagotomy in decerebrate cats. J. Physiol. 586 (17), 4265–4282. Errchidi, S., Hilaire, G., Monteau, R., 1990. Permanent release of noradrenalin modulates respiratory frequency in the newborn rat: an in vitro study. J. Physiol. (Lond.) 429, 497–510. Errichidi, S., Monteau, R., Hilaire, G., 1991. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study. J. Physiol. (Lond.) 443, 477–498. Fairchild, J., 1948. Effects of cold on infant rats: body temperatures, oxygen consumption, electrocardiograms. Am. J. Physiol. 155, 355–365. Fong, A.Y., Corcoran, A.E., Zimmer, M.B., Andrade, D.V., Milsom, W.K., 2008. Respiratory rhythm of brainstem-spinal cord preparations: effects of maturation, age, mass and oxygenation. Respir. Physiol. Neurobiol. 164, 429–440. Gajda, B.M., Fong, A.Y., Milsom, W.K., 2010. Species differences in respiratory rhythm generation in rodents. Am. J. Physiol. 298, 887–898. Hilaire, G., Duron, B., 1999. Maturation of the mammalian respiratory system. Physiol. Rev. 79 (2), 325–332. Hilaire, G., Monteau, R., Errchidi, S., 1989. Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat. Brain Res. 485, 325–332. Hilaire, G., Viemari, J.-C., Coulon, P., Simonneau, M., Bevengut, M., 2004. Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir. Physiol. Neurobiol. 143, 187–197. Hill, R.W., 2000. Anoxia tolerance to oxygen necessity: paradigm shift in the physiology of survival of apneic deep hypothermia in neonatal rodents. In: Heldmaier, G., Klingenspor, M. (Eds.), Life in the Cold, Eleventh International Hibernation Symposium. Springer, Heidelberg, Germany. pp. 199–205. Kieboom, J.K., Verkade, H.J., Burgerhof, J.G., Bierens, J.J., van Rheenen, P.F., Kneyber, M.C., Albers, M.J., 2015. Outcome after resuscitation beyond 30 minutes in drowned children with cardiac arrest and hypothermia: dutch nationwide retrospective cohort
Acknowledgement This work was funded by the NSERC of Canada (W.K. Milsom, 587150). References
Our study supports other studies that demonstrate that the brainstem-spinal cord preparation is not hypoxic with or without the pons (Fong et al., 2008; Murakoshi et al., 1985). Murakoshi et al. (1985) demonstrated that the change from a bell-shaped burst to a gasp-like decrementing burst pattern was due to the removal of sensory afferents and argued that it was not due to hypoxia. Fong et al., 2008 showed that the surface of the brainstem-spinal cord is hyperoxic and at the level of the preBötzinger complex, O2 levels were around 100 Torr. They also showed no differences in oxygen at three different positions relative to midline. The present study showed that the levels of oxygen were ∼40 Torr at the level of the preBötzinger complex at 27 °C, however during cooling the levels of O2 increased with the decreasing temperature. Our results show that there is an excess of oxygen in the brainstem at colder temperatures; further evidence that hypoxia and subsequent gasping are not involved in reinitiating the respiratory cycle during rewarming. This is not surprising since oxygen solubility increases as temperature decreases. Tissue oxygen levels could also be higher due to a decrease in cellular metabolism in the cold. Despite the increased oxygen supply in the brainstem regions, we observed a consistent fragmentation of the burst shape at the lowest temperatures during our stepwise cooling and rewarming. This was also observed by Mellen et al. (2002) but only during the transient rewarming phase in their experiments. Since they observed this irregular respiratory output from both field potentials around the preBötzinger complex and the phrenic motor output, they speculated that the fragmentation or irregular discharge originated from a lack of synchronization within the network in the medulla and not from disruption of the descending motor drive. 4.5. Conclusions Targeted temperature management for the treatment of acute brain injury is being widely used to enhance survival of neonates and children in clinical settings (Benedetti and Silverstein, 2018). Case studies show varying degrees of clinical outcomes after accidental hypothermia and drowning. A Dutch retrospective study showed that maintaining resuscitation efforts past 30 min results in poor outcomes in drowned victims with hypothermia and cardiac arrest (Kieboom et al. (2015). Understanding temperature effects on neural function and the specific developmental changes in respiratory function that occur during the early neonatal period are critical to help develop better therapeutic hypothermia strategies and to help determine when resuscitation efforts may be useful. Cooling neonatal rat brainstem–spinal cord preparations reduced respiratory frequency followed by respiratory arrest. Autoresuscitation 8
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