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Isoflurane exerts neuroprotective actions at or near the time of severe traumatic brain injury
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Isoflurane exerts neuroprotective actions at or near the time of severe traumatic brain injury Kimberly D. Statler a,d,⁎, Henry Alexander d,1 , Vincent Vagni d,1 , Richard Holubkov e,2 , C. Edward Dixon a,d,1 , Robert S.B. Clark a,d,1 , Larry Jenkins c,d,1 , Patrick M. Kochanek a,b,d,1 a
Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA c Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA d Safar Center for Resuscitation Research, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA e Department of Pediatrics, University of Utah School of Medicine, UT 84158, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Isoflurane improves outcome vs. fentanyl anesthesia, in experimental traumatic brain
Accepted 16 December 2005
injury (TBI). We assessed the temporal profile of isoflurane neuroprotection and tested
Available online 13 February 2006
whether isoflurane confers benefit at the time of TBI. Adult, male rats were randomized to isoflurane (1%) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h) for 30 min pre-TBI.
Keywords:
Anesthesia was discontinued, rats recovered to tail pinch, and TBI was delivered by
Anaesthesia
controlled cortical impact. Immediately post-TBI, rats were randomized to 1 h of isoflurane,
Traumatic brain injury
fentanyl, or no additional anesthesia, creating 6 anesthetic groups (isoflurane:isoflurane,
Fentanyl
isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, fentanyl:none).
Rat
Beam balance, beam walking, and Morris water maze (MWM) performances were assessed
Narcotic
over post-trauma d1-20. Contusion volume and hippocampal survival were assessed on d21.
Hippocampus
Rats receiving isoflurane pre- and post-TBI exhibited better beam walking and MWM
Head injury
performances than rats treated with fentanyl pre- and any treatment post-TBI. All rats
Contusion
pretreated with isoflurane had better CA3 neuronal survival than rats receiving fentanyl
Controlled cortical impact
pre- and post-TBI. In rats pretreated with fentanyl, post-traumatic isoflurane failed to affect function but improved CA3 neuronal survival vs. rats given fentanyl pre- and post-TBI. Posttraumatic isoflurane did not alter histopathological outcomes in rats pretreated with isoflurane. Rats receiving fentanyl pre- and post-TBI had the worst CA1 neuronal survival of all groups. Our data support isoflurane neuroprotection, even when used at the lowest feasible level before TBI (i.e., when discontinued with recovery to tail pinch immediately before injury). Investigators using isoflurane must consider its beneficial effects in the design and interpretation of experimental TBI research. © 2006 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Division of Critical Care Medicine, Department of Pediatrics, University of Utah School of Medicine, PO Box 581289, Salt Lake City, UT 84158, USA. Fax: +1 801 581 8686. E-mail addresses: [email protected] (K.D. Statler), [email protected] (H. Alexander), [email protected] (V. Vagni), [email protected] (R. Holubkov), [email protected] (C.E. Dixon), [email protected] (R.S.B. Clark), [email protected] (L. Jenkins), [email protected] (P.M. Kochanek). 1 Fax: +1 412 624 0943. 2 Intermountain Injury Control Center, University of Utah, PO Box 581289, Salt Lake City, UT 84158, USA. Fax: +1 801 581 8686. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.106
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1.
Introduction
Isoflurane, one of the most commonly used anesthetics in experimental traumatic brain injury (TBI), confers beneficial effects on functional outcomes compared to fentanyl anesthesia in experimental TBI (Statler et al., 2000, 2001, 2003, 2006). Specifically, in a direct comparison study, in which treatment with either isoflurane or the narcotic fentanyl was initiated before and continued 4 h after TBI, isoflurane-treated rats had better post-traumatic functional outcomes and less hippocampal neuronal death (Statler et al., 2000). A follow-up comprehensive comparison study of isoflurane and six clinically relevant agents (diazepam, fentanyl, ketamine, morphine, pentobarbital, and propofol) corroborated this finding, with isoflurane-treated rats demonstrating the quickest functional recovery and the best hippocampal neuronal survival after TBI (Statler et al., 2006). In order to mimic clinical TBI, in which victims are not anesthetized at the time of injury, as close as humanely possible, all experimental groups in the comprehensive comparison study had been treated with isoflurane prior to injury. TBI was delivered immediately upon return of tail pinch response, and the study drug was then administered immediately after TBI. Interestingly, rats receiving no sustained additional post-traumatic anesthesia (i.e., rats only pretreated with isoflurane) had similar outcomes to those administered additional post-traumatic isoflurane, suggesting that residual effects of pretreatment with isoflurane conferred benefit at the time of TBI (Statler et al., 2006). Further, the beneficial effects of isoflurane vs. fentanyl in our comprehensive comparison study (Statler et al., 2006) did not appear as marked as in our prior study, in which rats were injured while fully anesthetized with either isoflurane or fentanyl (Statler et al., 2000). Based on these observations, we speculated that putative neuroprotective effects of isoflurane anesthesia might be confounding results in experimental TBI, even when isoflurane is stopped immediately prior to TBI. The current study was designed to assess the temporal profile of the protective effects of isoflurane. We hypothesized that pretreatment with isoflurane exerts beneficial effects at the time of TBI. Further, we suspected that administration of additional posttraumatic isoflurane provides minimal added benefit. To test our hypotheses, we have systematically compared functional and histopathological outcomes in experimental TBI in adult, male Sprague–Dawley rats treated with either isoflurane or fentanyl for 30 min before TBI, followed by post-traumatic administration of 1 h of isoflurane, fentanyl, or no additional anesthesia. Our study design thus includes 6 pre:post-TBI anesthetic groups (isoflurane:isoflurane, isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, and fentanyl:none). Given the ubiquitous use of isoflurane in experimental TBI, our results will have direct implications for the design and interpretation of future investigations in experimental TBI.
2.
Results
In all groups, rectal and brain temperatures were controlled at 37 ± 0.5 °C, PaCO2 was maintained between 35 and 45 mm Hg,
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PaO2 was greater than 100 mm Hg, and hematocrit values remained within normal limits. Serum glucose values ranged between 140 and 250 mg/dl and did not differ between groups. MAP was higher in rats during treatment with fentanyl vs. isoflurane (P b 0.05, Fig. 1), but remained within the autoregulatory range for the adult rat in all groups (Hernandez et al., 1978; Hoffman et al., 1991). This effect on MAP was expected (Miura et al., 1999; Statler et al., 2000) and can be attributed to the vasodilatory actions of isoflurane. Since we were investigating whether pretreatment with isoflurane exerts beneficial effects at or near the time of TBI, we compared the durations of preparatory surgery using isoflurane anesthesia to assure that they were similar among experimental groups. This period was defined as the time from initiation of isoflurane anesthesia until completion of the craniotomy. Average (SEM) preparatory surgery times ranged from 25.7 (1.7) to 31.9 (2.7) min and did not differ among treatment groups (P = 0.258). The results of post-traumatic motor function tests are shown in Figs. 2 and 3. All injured groups showed impaired performances on beam walking tasks compared to shams (P b 0.05, Fig. 2). Additionally, rats treated with fentanyl: isoflurane, fentanyl:fentanyl, or fentanyl:none had longer beam walking latencies compared to rats treated with isoflurane:isoflurane (P b 0.05). Conversely, latencies were similar between rats treated with isoflurane:isoflurane, isoflurane:fentanyl, or isoflurane:none. Performances on beam balance tasks were similar among all groups (Fig. 3). Cognitive outcomes, assessed 14–18 days after injury using a submerged platform paradigm of the MWM, are shown in Fig. 4. All injured groups had impaired MWM performances compared to shams (P b 0.05). Additionally, rats treated with fentanyl:isoflurane, fentanyl:fentanyl, or fentanyl:none showed longer latencies to find the hidden platform than rats treated with isoflurane:isoflurane (P b 0.05). Conversely, performances were similar between rats treated with isoflurane:isoflurane, isoflurane:fentanyl, or isoflurane:none. Swim
Fig. 1 – Mean arterial pressure (MAP) vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane and fent indicates fentanyl. MAP is higher during treatment with fentanyl compared to isoflurane (*P b 0.05 vs. iso:iso or fent:fent, ^P b 0.05 vs. iso: fent, #P b 0.05 vs. fent:iso).
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Fig. 2 – Beam walking performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. All injured groups had impaired beam walking performances compared to shams (*P b 0.05 vs. sham). Regardless of post-traumatic therapy, groups pretreated with fentanyl took longer to escape an adverse stimulus of white noise than those treated with isoflurane pre- and post-TBI (#P b 0.05 vs. iso:iso). Post-traumatic treatment did not alter performances among groups pretreated with fentanyl. Likewise, performances are similar among groups pretreated with isoflurane, regardless of post-traumatic treatment.
speeds were similar in all experimental groups, indicating that the observed differences were not due to residual motor deficits. Lesion volumes, assessed 21 days after TBI, were similar between experimental groups (Fig. 5A). In contrast, rats treated with fentanyl:fentanyl had more CA3 hippocampal death than rats treated with isoflurane:isoflurane, isoflurane: fentanyl, isoflurane:none, or fentanyl:isoflurane (Fig. 5B, P b 0.05). Similarly, rats treated with fentanyl:fentanyl had more CA1 hippocampal death than in any other experimental group (Fig. 5C, P b 0.05 vs. isoflurane:isoflurane, isoflurane:
fentanyl, isoflurane:none, fentanyl:isoflurane, or fentanyl: none).
3.
Discussion
Motor and cognitive outcomes after TBI were minimally affected by the post-traumatic treatment and largely determined by anesthetic administration immediately prior to TBI. Rats pretreated with fentanyl, followed by any post-traumatic treatment, had worse beam walking and MWM performances
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Fig. 3 – Beam balance performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. Beam balance performances are similar among all injured groups and shams.
than rats treated with isoflurane pre- and post-TBI. Importantly, post-traumatic administration of isoflurane failed to improve functional or cognitive outcome in rats administered fentanyl before TBI. Likewise, functional and cognitive outcomes were similar among all experimental groups pretreated with isoflurane, indicating that administration of additional post-traumatic isoflurane provided minimal added benefit to functional outcome compared to isoflurane pretreatment alone. Similarly, the poorest histopathological outcomes were observed in rats treated with fentanyl pre- and post-TBI. CA3 neuronal survival was better in rats pretreated with isoflurane, followed by any post-traumatic treatment, compared to rats receiving fentanyl pre- and post-TBI. Although posttraumatic treatment with isoflurane did not improve functional outcomes, it did provide histopathological protection in rats pretreated with fentanyl, improving CA3 neuronal
survival compared to the fentanyl:fentanyl treatment group. Conversely, administration of additional post-traumatic isoflurane failed to alter hippocampal neuronal survival in rats pretreated with isoflurane, suggesting that additional posttraumatic isoflurane provides minimal added histopathological benefit over isoflurane even when administration is limited to the period immediately prior to TBI. Our results support the hypotheses that residual effects of pretreatment with isoflurane are highly beneficial at the time of TBI and that administration of additional post-traumatic isoflurane provides minimal added benefit. The neuroprotective actions of isoflurane are likely multifactorial and may be attributed, in part, to cerebral vasodilation and attenuation of excitotoxicity. Isoflurane is a cerebral blood flow promoter (Hendrich et al., 2001; Lenz et al., 1998) and may attenuate post-traumatic hypoperfusion. Additionally, isoflurane has been shown to reduce glutamate release (Patel et al., 1993),
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Fig. 4 – Morris water maze (MWM) performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. All injured groups have longer latencies to find a submerged platform in the MWM compared to shams (*P b 0.05 vs. sham). Regardless of post-traumatic therapy, all groups pretreated with fentanyl have worse performances than those given isoflurane pre- and post-TBI (#P b 0.05 vs. iso:iso). Performances do not differ among groups pretreated with fentanyl, regardless of post-traumatic therapy. Similarly, performances are similar among all groups pretreated with isoflurane.
block NMDA receptors (Bickler et al., 1994), and reduce NMDAmediated calcium influx (Bickler et al., 1994). In the controlled cortical impact model, post-traumatic hyperglycolysis is attenuated by isoflurane compared to fentanyl (Statler et al., 2003). Finally, in cerebral ischemia models, GABAA-agonist actions of isoflurane are neuroprotective (Bickler et al., 2003). An alternative explanation for our results is that fentanyl anesthesia is directly detrimental in experimental TBI. Endogenous opioids have been implicated in the mediation of secondary injury after TBI (McIntosh et al., 1987). Putative deleterious effects of fentanyl may include induced subcortical neuronal excitation (Kofke et al., 1996; Safo et al., 1985; Tempelhoff et al., 1992) and reduction of cerebral blood flow
(Nikolaishvili et al., 2004; Safo et al., 1985). In both rats and humans, high doses of fentanyl, which acts as a mu-agonist, may promote neuronal excitability and cause subcortical seizures (Kofke et al., 1996; Safo et al., 1985; Tempelhoff et al., 1992). At doses similar to those used in our study, fentanyl has been shown to decrease cerebral blood flow in rats (Nikolaishvili et al., 2004; Safo et al., 1985), potentially exacerbating post-traumatic hypoperfusion. However, endogenous opioids may also be important for endogenous neuroprotection (Hayes et al., 1990), and other investigators have reported that fentanyl anesthesia does not exacerbate cerebral injury (Soonthon-Brant et al., 1999). Our recent comprehensive comparison of seven anesthetic agents (diazepam, fentanyl,
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Fig. 5 – Histological outcomes 21 days after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. Hippocampal neuronal survival is expressed as neurons per high power field (h.p.f.). (A) Lesion volume, depicted in cubic mm, is similar among all injured groups. (B) CA3 hippocampal neuronal survival is worse in rats treated with fentanyl pre- and post-TBI, compared to any group pretreated with isoflurane (*P b 0.05 vs. iso:iso, iso:fent, iso:none). Post-traumatic treatment with isoflurane attenuates CA3 hippocampal neuronal death following fentanyl pretreatment (#P b 0.05 vs. fent:iso). (C) CA1 hippocampal neuronal death is worst in rats given fentanyl before and after TBI (†P b 0.05 vs. all other groups).
isoflurane, ketamine, morphine, pentobarbital, and propofol) in experimental TBI corroborates these findings. While we found that isoflurane-treated rats exhibited the best cognitive recovery and hippocampal survival, rats treated with fentanyl had outcomes similar to other anesthetic groups (Statler et al., 2000, 2001, 2003, 2006). Our findings suggest that, rather than being directly detrimental, fentanyl fails to afford neuroprotection (compared to isoflurane) in the controlled cortical impact model of experimental TBI. Further investigations, specifically focused on the interplay between fentanyl anes-
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thesia, secondary injury cascades, and neuronal excitation, are needed to further clarify the putative deleterious actions of fentanyl anesthesia in TBI. Recent work in cerebral ischemia has questioned the permanence of isoflurane neuroprotection (Elsersy et al., 2004; Kawaguchi et al., 2000, 2004). In these studies, early beneficial effects of isoflurane compared to fentanyl-treated (Kawaguchi et al., 2000, 2004) or awake (Elsersy et al., 2004) rats have dissipated by 2–3 weeks after the ischemic insult. In our study, isoflurane administration is associated with improved cognitive and histological outcomes 2–3 weeks after TBI, suggesting more long-lasting effects. Additional longitudinal studies are needed to more clearly delineate the long-term effects of isoflurane in experimental TBI. While the permanence of isoflurane neuroprotection remains unclear, given the widespread use of isoflurane anesthesia in experimental TBI, even transient benefits conferred by isoflurane may have important implications for study design and interpretation. It is interesting that post-traumatic isoflurane attenuated hippocampal neuronal death in rats treated with fentanyl just prior to TBI, yet failed to alter functional outcome. These findings may reflect a lack of correlation between the effects of isoflurane on functional and histopathological parameters. Other investigators have reported similar discrepancies between functional and histopathological outcomes after TBI (Clark et al., 2000; Varma et al., 2002; Whalen et al., 1999). Alternatively, isoflurane may promote subtle improvements in motor or cognitive performance. The functional and cognitive testing methods applied in our study may lack sufficient sensitivity to detect such subtle differences. Indeed, in clinical TBI studies, more intricate and complex tasks are often necessary to unmask post-traumatic functional deficits or improvements (Chapman et al., 2000). More complex testing batteries may similarly be necessary to reveal subtle differences between treatment groups in experimental TBI. Thus, the use of relatively simple (albeit standard) functional and cognitive outcome tests may be a limitation of our study. Hippocampal neuronal survival was assessed in our study using manual hippocampal neuronal counts in a single 10-μm coronal brain section taken 5-mm from the occiput and stained with cersyl violet. Cresyl violet does not stain neurons specifically. Surviving neurons were distinguished from glia based on cell morphology and size. We acknowledge that this assessment technique is not as rigorous as the use of neuronspecific staining techniques or classical stereological techniques. This is a limitation of our study. Further, more rigorous histopathological assessment is needed to better clarify the histological protection afforded by isoflurane in our study. The comparison of inhalational and intravenous anesthetic agents is another limitation of our study. It is difficult to compare anesthetic depth, as defined by the classic concept of minimal alveolar concentration (MAC), between these two techniques. We chose dosing regimens based on literature review (Miura et al., 1999; Soonthon-Brant et al., 1999) and pilot studies in our laboratory. The doses chosen are identical to those used in our prior investigations (Statler et al., 2000, 2003, 2006). Although no experimental group demonstrated signs of increased discomfort or stress, such as unexplained hypertension or increased hyperglycemia, equivalent anesthesia, as defined by the classic concept of MAC, may not have
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been achieved. Consequently, our results may reflect differences in the post-traumatic stress response, as well as differences in anesthetic actions. Nonetheless, we have compared dosing regimens of isoflurane and fentanyl that are commonly applied in experimental brain injury models and shown that rats treated with fentanyl prior to TBI showed worse outcomes. We have shown that isoflurane, a commonly used anesthetic in experimental TBI, confers beneficial effects even when administered at the lowest feasible level before TBI (i.e., when discontinued with recovery to tail pinch immediately before injury). In addition, administration of post-traumatic isoflurane produces benefits only in rats initially anesthetized with fentanyl and only on histopathology. Our results indicate that isoflurane exerts neuroprotective actions at or very near the time of TBI. Since it is inhumane to subject an awake animal to TBI, our findings have direct implications for study design and interpretation in experimental TBI. We are not advocating that use of isoflurane be abandoned; however, investigators using isoflurane must be cognizant of and give careful consideration to the beneficial effects of this anesthetic. Myriad factors dictate anesthetic choice in experimental TBI, and these clearly differ among studies. Investigators should weigh the effects of different anesthetic agents and tailor their choice accordingly. Although isoflurane is easy to use and commonly preferred, at times use of a less neuroprotective anesthetic, such as fentanyl, may be preferable.
4.
Experimental procedures
Adult, male (300–440 g), Sprague–Dawley rats were used in all experiments. The rats were allowed free access to food and water before and after surgery. The University of Pittsburgh Animal Care and Use Committee approved all studies. All surgical procedures were performed using aseptic technique. Rats were anesthetized with 4% isoflurane (Isoflo, Abbott Laboratories, North Chicago, IL) via nose cone and then endotracheally intubated with a 14-guage angiocatheter and mechanically ventilated. Anesthesia was maintained with 2–2.5% isoflurane for the duration of surgical preparation. Femoral vessels were cannulated for continuous arterial blood pressure monitoring, blood sampling, and medication administration. Pancuronium bromide (0.1 mg/kg/h, ElkinsSinn, Cherry Hill, NJ) was given for muscle relaxation. Core temperature was monitored via a rectal probe and controlled at 37 ± 0.5 °C. The rat was placed into a stereotaxic frame (David Kopf, Tujunga, CA) and a craniotomy (6 × 6mm) was performed over the left parietal cortex using a high-speed dental drill. The dura and bone flap were left in place until immediately before CCI. A temperature probe (2.28-mm outside diameter, Physitemp Corp., Clifton, NJ) was placed through a burr hole into the left frontal lobe, and brain temperature was controlled at 37 ± 0.5 °C. At the end of preparatory surgery, rats were randomized (n = 27 per group) to 30 min of either isoflurane or fentanyl before TBI. In the isoflurane group, isoflurane was continued at 1%. In the fentanyl group, isoflurane was stopped and fentanyl (50 mcg/ml, Elkins-Sinn, Cherry Hill, NJ) was
administered as a 10 mcg/kg iv bolus followed by a 50 mcg/kg/h iv infusion. In order to mimic clinical TBI, in which victims are not anesthetized at the time of injury, as close as humanely possible, either isoflurane or fentanyl was stopped at the end of the 30 min equilibration period. Rats were allowed to recover to tail pinch response and controlled cortical impact (6-mm tip, 4 m/s velocity, 1.8-mm depth of deformation) was performed immediately before rats had fully awakened from anesthesia. This anesthetic approach has been used in other TBI studies (Jenkins et al., 2002; Statler et al., 2006) and represents the lowest feasible, humane level of anesthesia at the time of TBI. Immediately after TBI, rats were randomized (n = 9 per group) to 1 h of isoflurane (1% by inhalation), fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h), or no additional anesthesia. (In order to facilitate humane closure of the craniotomy and skin incision, isoflurane 1% was administered for approximately 5 min post-TBI to rats in the no additional anesthesia groups.) The 1-h post-traumatic treatment period was chosen based on the initial posttraumatic period of maximal excitotoxicity (Palmer et al., 1993). Allowing rats to awaken immediately after TBI (i.e., administering no additional post-traumatic anesthesia) corresponds to the standard anesthetic approach used by several laboratories (Hallam et al., 2004; Kozlowski et al., 2004), including ours (Kochanek et al., 1995). This study design created six anesthetic groups (n = 9 per group), based on pre:post-TBI anesthesia: isoflurane:isoflurane, isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, and fentanyl:none. As an additional control, shams underwent craniotomy using isoflurane anesthesia with recovery to tail pinch, but no TBI or additional anesthesia. At the end of the 1-h treatment period, rats were allowed to awaken and recovered to extubation. Mean arterial blood pressure (MAP) and rectal and brain temperatures were monitored continuously during the 1h treatment period. Blood glucose levels, arterial blood gas samples, and hematocrit were assessed every 15 min. PaCO2 was controlled at 35–45 mm Hg, and PaO2 was greater than 100 mm Hg throughout the experiment in all preparations. Motor function was assessed by beam walking and beam balance tasks on days 1–5 after injury by an observer masked to experimental group. Cognitive outcome was assessed using a submerged-platform paradigm of the Morris water maze (MWM) on days 14–18 after injury by an observer masked to experimental group. On postinjury day 21, rats were reanesthetized with isoflurane 4% and perfused with heparinized saline followed by 4% paraformaldehyde. Brains were removed, postfixed, and cryoprotected. Serial coronal sections (10-μm) were made at 1-mm intervals throughout the brain, mounted on slides, and stained with cresyl violet. Lesion volume was assessed by an observer masked to experimental group, using an image analysis system (MCID, Imaging Research, St. Catherine's, Ontario, Canada). Surviving hippocampal neurons, identified by morphology and size, were counted by an observer masked to experimental group, under ×400 magnification in the anatomic CA1 and CA3
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hippocampal regions in a single 10-μm coronal section taken 5-mm from the occiput.
4.1.
Statistical analysis
All statistical analyses were performed using either SPSS, version 11 for MAC OS X or SAS, version 9.1 for Windows. Arterial blood gas values, blood glucose levels, and MAP were compared using two-way analysis of variance for repeated measures. Beam balance and beam walking results and MWM latencies were compared using a linear mixed model with experimental group and time-specified as categorical main effects. An unstructured covariance matrix was used to model the association between measures for each subject over time. The duration of preparatory surgery, lesion volume, and hippocampal neuronal survival were compared using one-way analysis of variance. Tukey's test was used in all post hoc comparisons, where appropriate. A P b 0.05 was considered significant. Data are presented as mean ± SEM.
Acknowledgments We thank the U.S. Army (DMAD 17-91-7009), the National Institutes of Health (NS30318 and T32-HD40686), and the Laerdal Foundation for Acute Medicine for generous support of this project. We also thank Marci Provins for assistance with preparation of the manuscript.
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perfusion during anesthesia with fentanyl, isoflurane, or pentobarbital in normal rats studied by arterial spin-labeled MRI. Magn. Reson. Med. 46, 2002–2006. Hernandez, M.J., Brennan, R.W., Bowman, G.S., 1978. Cerebral blood flow autoregulation in the rat. Stroke 9, 150–154. Hoffman, W.E., Edelman, G., Kochs, E., Werner, C., Segil, L., Albrecht, R.F., 1991. Cerebral autoregulation in awake versus isoflurane-anesthetized rats. Anesth. Analg. 73, 753–757. Jenkins, L.W., Peters, G.W., Dixon, C.E., Zhang, X., Clark, R.S., Skinner, J.C., Marion, D.W., Adelson, P.D., Kochanek, P.M., 2002. Conventional and functional proteomics using large format two-dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats. J. Neurotrauma 19, 715–740. Kawaguchi, M., Kimbro, J.R., Drummond, J.C., Cole, D.J., Kelly, P.J., Patel, P.M., 2000. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 92, 1335–1342. Kawaguchi, M., Drummond, J.C., Cole, D.J., Kelly, P.J., Spurlock, M.P., Patel, P.M., 2004. Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia. Anesth. Analg. 98, 798–805. Kochanek, P.M., Marion, D., Zhang, W., Schiding, J.K., White, M., Palmer, A., Clark, R.S.B., O'Mally, M., Styren, S., Ho, C., DeKosky, S., 1995. Severe controlled cortical impact in rats: assessment of cerebral edema, blood flow, and contusion volume. J. Neurotrauma 12, 1015–1025. Kofke, W.A., Garman, R.H., Janosky, J., Rose, M.E., 1996. Opioid neurotoxicity: neuropathologic effects in rats of different fentanyl congeners and the effects of hexamethoniuminduced normotension. Anesth. Analg. 83, 141–146. Kozlowski, D.A., Nahed, B.V., Hovda, D.A., Lee, S.M., 2004. Paradoxical effects of cortical impact injury on environmentally enriched rats. J. Neurotrauma 21, 513–519. Lenz, C., Rebel, A., van Ackern, K., Kuschinsky, W., Waschke, K.F., 1998. Local cerebral blood flow, local cerebral glucose utilization, and flow-metabolism coupling during sevoflurane versus isoflurane anesthesia in rats. Anesthesiology 89, 1480–1488. McIntosh, T.K., Hayes, R.L., DeWitt, D.S., Agura, V., Faden, A.I., 1987. Endogenous opioids may mediate secondary damage after experimental brain injury. Am. J. Physiol. 253, E565–E674. Miura, Y., Mackensen, G.B., Nellgard, B., Pearlstein, R.D., Bart, R.D., Dexter, F., Warner, D.S., 1999. Effects of isoflurane, ketamine, and fentanyl/N2O on concentrations of brain and plasma catecholamines during near-complete cerebral ischemia in the rat. Anesth. Analg. 88, 787–792. Nikolaishvili, L.S., Gobechiya, L.S., Mitagvariya, N.P., 2004. The effects of fentanyl and morphine on local blood flow and oxygen tension in the frontoparietal cortex and nucleus accumbens of the brain in white rats. Neurosci. Behav. Physiol. 34, 467–471. Palmer, A.M., Marion, D.W., Botscheller, M.L., Swedlow, P.E., Styren, S.D., DeKosky, S.T., 1993. Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J. Neurochem. 61, 2015–2024. Patel, P.M., Drummond, J.C., Goskowicz, R., Sano, T., Cole, D.J., 1993. The volatile anesthetic isoflurane reduces ischemia induced release of glutamate in rats. J. Cereb. Blood Flow Metab. 1993, S685. Safo, Y., Young, M.L., Greenberg, J., Carlsson, C., Reivich, M., Keykhah, M., Harp, J.R., 1985. Effects of fentanyl on local cerebral blood flow in the rat. Acta Anaesthesiol. Scand. 29, 594–598. Soonthon-Brant, V., Patel, P.M., Drummond, J.C., Cole, D.J., Kelly, P.J., Watson, M., 1999. Fentanyl does not increase brain injury after focal cerebral ischemia in rats. Anesth. Analg. 88, 49–55. Statler, K.D., Kochanek, P.M., Dixon, C.E., Alexander, H.L., Warner, D.S., Clark, R.S., Wisniewski, S.R., Graham, S.H., Jenkins, L.W.,
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Marion, D.W., Safar, P.J., 2000. Isoflurane improves long-term neurologic outcome versus fentanyl after traumatic brain injury in rats. J. Neurotrauma 17, 1179–1189. Statler, K.D., Jenkins, L.W., Dixon, C.E., Clark, R.S., Marion, D.W., Kochanek, P.M., 2001. The simple model versus the super model: translating experimental traumatic brain injury research to the bedside. J. Neurotrauma 18, 1195–1206. Statler, K.D., Janesko, K.L., Melick, J.A., Clark, R.S., Jenkins, L.W., Kochanek, P.M., 2003. Hyperglycolysis is exacerbated after traumatic brain injury with fentanyl vs. isoflurane anesthesia in rats. Brain Res. 994, 37–43. Statler, K.D., Alexander, H., Vagni, V., Dixon, C., Clark, R., Jenkins, L., Kochanek, P., 2006. Comparison of seven anesthetic agents on outcome after experimental traumatic brain injury in adult, male rats. J. Neurotrauma 23 (1).
Tempelhoff, R., Modica, P.A., Bernardo, K.L., Edwards, I., 1992. Fentanyl-induced electrocorticographic seizures in patients with complex partial epilepsy. J. Neurosurg. 77, 201–208. Varma, M.R., Dixon, C.E., Jackson, E.K., Peters, G.W., Melick, J.A., Griffith, R.P., Vagni, V.A., Clark, R.S., Jenkins, L.W., Kochanek, P.M., 2002. Administration of adenosine receptor agonists or antagonists after controlled cortical impact in mice: effects on function and histopathology. Brain Res. 951, 191–201. Whalen, M.J., Clark, R.S., Dixon, C.E., Robichaud, P., Marion, D.W., Vagni, V., Graham, S.H., Virag, L., Hasko, G., Stachlewitz, R., Szabo, C., Kochanek, P.M., 1999. Reduction of cognitive and motor deficits after traumatic brain injury in mice deficient in poly(ADP-ribose) polymerase. J. Cereb. Blood Flow Metab. 19, 835–842.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Isoflurane exerts neuroprotective actions at or near the time of severe traumatic brain injury Kimberly D. Statler a,d,⁎, Henry Alexander d,1 , Vincent Vagni d,1 , Richard Holubkov e,2 , C. Edward Dixon a,d,1 , Robert S.B. Clark a,d,1 , Larry Jenkins c,d,1 , Patrick M. Kochanek a,b,d,1 a
Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA c Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA d Safar Center for Resuscitation Research, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA e Department of Pediatrics, University of Utah School of Medicine, UT 84158, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Isoflurane improves outcome vs. fentanyl anesthesia, in experimental traumatic brain
Accepted 16 December 2005
injury (TBI). We assessed the temporal profile of isoflurane neuroprotection and tested
Available online 13 February 2006
whether isoflurane confers benefit at the time of TBI. Adult, male rats were randomized to isoflurane (1%) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h) for 30 min pre-TBI.
Keywords:
Anesthesia was discontinued, rats recovered to tail pinch, and TBI was delivered by
Anaesthesia
controlled cortical impact. Immediately post-TBI, rats were randomized to 1 h of isoflurane,
Traumatic brain injury
fentanyl, or no additional anesthesia, creating 6 anesthetic groups (isoflurane:isoflurane,
Fentanyl
isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, fentanyl:none).
Rat
Beam balance, beam walking, and Morris water maze (MWM) performances were assessed
Narcotic
over post-trauma d1-20. Contusion volume and hippocampal survival were assessed on d21.
Hippocampus
Rats receiving isoflurane pre- and post-TBI exhibited better beam walking and MWM
Head injury
performances than rats treated with fentanyl pre- and any treatment post-TBI. All rats
Contusion
pretreated with isoflurane had better CA3 neuronal survival than rats receiving fentanyl
Controlled cortical impact
pre- and post-TBI. In rats pretreated with fentanyl, post-traumatic isoflurane failed to affect function but improved CA3 neuronal survival vs. rats given fentanyl pre- and post-TBI. Posttraumatic isoflurane did not alter histopathological outcomes in rats pretreated with isoflurane. Rats receiving fentanyl pre- and post-TBI had the worst CA1 neuronal survival of all groups. Our data support isoflurane neuroprotection, even when used at the lowest feasible level before TBI (i.e., when discontinued with recovery to tail pinch immediately before injury). Investigators using isoflurane must consider its beneficial effects in the design and interpretation of experimental TBI research. © 2006 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Division of Critical Care Medicine, Department of Pediatrics, University of Utah School of Medicine, PO Box 581289, Salt Lake City, UT 84158, USA. Fax: +1 801 581 8686. E-mail addresses: [email protected] (K.D. Statler), [email protected] (H. Alexander), [email protected] (V. Vagni), [email protected] (R. Holubkov), [email protected] (C.E. Dixon), [email protected] (R.S.B. Clark), [email protected] (L. Jenkins), [email protected] (P.M. Kochanek). 1 Fax: +1 412 624 0943. 2 Intermountain Injury Control Center, University of Utah, PO Box 581289, Salt Lake City, UT 84158, USA. Fax: +1 801 581 8686. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.106
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1.
Introduction
Isoflurane, one of the most commonly used anesthetics in experimental traumatic brain injury (TBI), confers beneficial effects on functional outcomes compared to fentanyl anesthesia in experimental TBI (Statler et al., 2000, 2001, 2003, 2006). Specifically, in a direct comparison study, in which treatment with either isoflurane or the narcotic fentanyl was initiated before and continued 4 h after TBI, isoflurane-treated rats had better post-traumatic functional outcomes and less hippocampal neuronal death (Statler et al., 2000). A follow-up comprehensive comparison study of isoflurane and six clinically relevant agents (diazepam, fentanyl, ketamine, morphine, pentobarbital, and propofol) corroborated this finding, with isoflurane-treated rats demonstrating the quickest functional recovery and the best hippocampal neuronal survival after TBI (Statler et al., 2006). In order to mimic clinical TBI, in which victims are not anesthetized at the time of injury, as close as humanely possible, all experimental groups in the comprehensive comparison study had been treated with isoflurane prior to injury. TBI was delivered immediately upon return of tail pinch response, and the study drug was then administered immediately after TBI. Interestingly, rats receiving no sustained additional post-traumatic anesthesia (i.e., rats only pretreated with isoflurane) had similar outcomes to those administered additional post-traumatic isoflurane, suggesting that residual effects of pretreatment with isoflurane conferred benefit at the time of TBI (Statler et al., 2006). Further, the beneficial effects of isoflurane vs. fentanyl in our comprehensive comparison study (Statler et al., 2006) did not appear as marked as in our prior study, in which rats were injured while fully anesthetized with either isoflurane or fentanyl (Statler et al., 2000). Based on these observations, we speculated that putative neuroprotective effects of isoflurane anesthesia might be confounding results in experimental TBI, even when isoflurane is stopped immediately prior to TBI. The current study was designed to assess the temporal profile of the protective effects of isoflurane. We hypothesized that pretreatment with isoflurane exerts beneficial effects at the time of TBI. Further, we suspected that administration of additional posttraumatic isoflurane provides minimal added benefit. To test our hypotheses, we have systematically compared functional and histopathological outcomes in experimental TBI in adult, male Sprague–Dawley rats treated with either isoflurane or fentanyl for 30 min before TBI, followed by post-traumatic administration of 1 h of isoflurane, fentanyl, or no additional anesthesia. Our study design thus includes 6 pre:post-TBI anesthetic groups (isoflurane:isoflurane, isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, and fentanyl:none). Given the ubiquitous use of isoflurane in experimental TBI, our results will have direct implications for the design and interpretation of future investigations in experimental TBI.
2.
Results
In all groups, rectal and brain temperatures were controlled at 37 ± 0.5 °C, PaCO2 was maintained between 35 and 45 mm Hg,
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PaO2 was greater than 100 mm Hg, and hematocrit values remained within normal limits. Serum glucose values ranged between 140 and 250 mg/dl and did not differ between groups. MAP was higher in rats during treatment with fentanyl vs. isoflurane (P b 0.05, Fig. 1), but remained within the autoregulatory range for the adult rat in all groups (Hernandez et al., 1978; Hoffman et al., 1991). This effect on MAP was expected (Miura et al., 1999; Statler et al., 2000) and can be attributed to the vasodilatory actions of isoflurane. Since we were investigating whether pretreatment with isoflurane exerts beneficial effects at or near the time of TBI, we compared the durations of preparatory surgery using isoflurane anesthesia to assure that they were similar among experimental groups. This period was defined as the time from initiation of isoflurane anesthesia until completion of the craniotomy. Average (SEM) preparatory surgery times ranged from 25.7 (1.7) to 31.9 (2.7) min and did not differ among treatment groups (P = 0.258). The results of post-traumatic motor function tests are shown in Figs. 2 and 3. All injured groups showed impaired performances on beam walking tasks compared to shams (P b 0.05, Fig. 2). Additionally, rats treated with fentanyl: isoflurane, fentanyl:fentanyl, or fentanyl:none had longer beam walking latencies compared to rats treated with isoflurane:isoflurane (P b 0.05). Conversely, latencies were similar between rats treated with isoflurane:isoflurane, isoflurane:fentanyl, or isoflurane:none. Performances on beam balance tasks were similar among all groups (Fig. 3). Cognitive outcomes, assessed 14–18 days after injury using a submerged platform paradigm of the MWM, are shown in Fig. 4. All injured groups had impaired MWM performances compared to shams (P b 0.05). Additionally, rats treated with fentanyl:isoflurane, fentanyl:fentanyl, or fentanyl:none showed longer latencies to find the hidden platform than rats treated with isoflurane:isoflurane (P b 0.05). Conversely, performances were similar between rats treated with isoflurane:isoflurane, isoflurane:fentanyl, or isoflurane:none. Swim
Fig. 1 – Mean arterial pressure (MAP) vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane and fent indicates fentanyl. MAP is higher during treatment with fentanyl compared to isoflurane (*P b 0.05 vs. iso:iso or fent:fent, ^P b 0.05 vs. iso: fent, #P b 0.05 vs. fent:iso).
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Fig. 2 – Beam walking performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. All injured groups had impaired beam walking performances compared to shams (*P b 0.05 vs. sham). Regardless of post-traumatic therapy, groups pretreated with fentanyl took longer to escape an adverse stimulus of white noise than those treated with isoflurane pre- and post-TBI (#P b 0.05 vs. iso:iso). Post-traumatic treatment did not alter performances among groups pretreated with fentanyl. Likewise, performances are similar among groups pretreated with isoflurane, regardless of post-traumatic treatment.
speeds were similar in all experimental groups, indicating that the observed differences were not due to residual motor deficits. Lesion volumes, assessed 21 days after TBI, were similar between experimental groups (Fig. 5A). In contrast, rats treated with fentanyl:fentanyl had more CA3 hippocampal death than rats treated with isoflurane:isoflurane, isoflurane: fentanyl, isoflurane:none, or fentanyl:isoflurane (Fig. 5B, P b 0.05). Similarly, rats treated with fentanyl:fentanyl had more CA1 hippocampal death than in any other experimental group (Fig. 5C, P b 0.05 vs. isoflurane:isoflurane, isoflurane:
fentanyl, isoflurane:none, fentanyl:isoflurane, or fentanyl: none).
3.
Discussion
Motor and cognitive outcomes after TBI were minimally affected by the post-traumatic treatment and largely determined by anesthetic administration immediately prior to TBI. Rats pretreated with fentanyl, followed by any post-traumatic treatment, had worse beam walking and MWM performances
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Fig. 3 – Beam balance performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. Beam balance performances are similar among all injured groups and shams.
than rats treated with isoflurane pre- and post-TBI. Importantly, post-traumatic administration of isoflurane failed to improve functional or cognitive outcome in rats administered fentanyl before TBI. Likewise, functional and cognitive outcomes were similar among all experimental groups pretreated with isoflurane, indicating that administration of additional post-traumatic isoflurane provided minimal added benefit to functional outcome compared to isoflurane pretreatment alone. Similarly, the poorest histopathological outcomes were observed in rats treated with fentanyl pre- and post-TBI. CA3 neuronal survival was better in rats pretreated with isoflurane, followed by any post-traumatic treatment, compared to rats receiving fentanyl pre- and post-TBI. Although posttraumatic treatment with isoflurane did not improve functional outcomes, it did provide histopathological protection in rats pretreated with fentanyl, improving CA3 neuronal
survival compared to the fentanyl:fentanyl treatment group. Conversely, administration of additional post-traumatic isoflurane failed to alter hippocampal neuronal survival in rats pretreated with isoflurane, suggesting that additional posttraumatic isoflurane provides minimal added histopathological benefit over isoflurane even when administration is limited to the period immediately prior to TBI. Our results support the hypotheses that residual effects of pretreatment with isoflurane are highly beneficial at the time of TBI and that administration of additional post-traumatic isoflurane provides minimal added benefit. The neuroprotective actions of isoflurane are likely multifactorial and may be attributed, in part, to cerebral vasodilation and attenuation of excitotoxicity. Isoflurane is a cerebral blood flow promoter (Hendrich et al., 2001; Lenz et al., 1998) and may attenuate post-traumatic hypoperfusion. Additionally, isoflurane has been shown to reduce glutamate release (Patel et al., 1993),
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Fig. 4 – Morris water maze (MWM) performance vs. time after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. All injured groups have longer latencies to find a submerged platform in the MWM compared to shams (*P b 0.05 vs. sham). Regardless of post-traumatic therapy, all groups pretreated with fentanyl have worse performances than those given isoflurane pre- and post-TBI (#P b 0.05 vs. iso:iso). Performances do not differ among groups pretreated with fentanyl, regardless of post-traumatic therapy. Similarly, performances are similar among all groups pretreated with isoflurane.
block NMDA receptors (Bickler et al., 1994), and reduce NMDAmediated calcium influx (Bickler et al., 1994). In the controlled cortical impact model, post-traumatic hyperglycolysis is attenuated by isoflurane compared to fentanyl (Statler et al., 2003). Finally, in cerebral ischemia models, GABAA-agonist actions of isoflurane are neuroprotective (Bickler et al., 2003). An alternative explanation for our results is that fentanyl anesthesia is directly detrimental in experimental TBI. Endogenous opioids have been implicated in the mediation of secondary injury after TBI (McIntosh et al., 1987). Putative deleterious effects of fentanyl may include induced subcortical neuronal excitation (Kofke et al., 1996; Safo et al., 1985; Tempelhoff et al., 1992) and reduction of cerebral blood flow
(Nikolaishvili et al., 2004; Safo et al., 1985). In both rats and humans, high doses of fentanyl, which acts as a mu-agonist, may promote neuronal excitability and cause subcortical seizures (Kofke et al., 1996; Safo et al., 1985; Tempelhoff et al., 1992). At doses similar to those used in our study, fentanyl has been shown to decrease cerebral blood flow in rats (Nikolaishvili et al., 2004; Safo et al., 1985), potentially exacerbating post-traumatic hypoperfusion. However, endogenous opioids may also be important for endogenous neuroprotection (Hayes et al., 1990), and other investigators have reported that fentanyl anesthesia does not exacerbate cerebral injury (Soonthon-Brant et al., 1999). Our recent comprehensive comparison of seven anesthetic agents (diazepam, fentanyl,
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Fig. 5 – Histological outcomes 21 days after injury. Treatment groups are indicated as pre:post-TBI treatment such that iso indicates isoflurane, fent indicates fentanyl, and none indicates no additional anesthesia. Hippocampal neuronal survival is expressed as neurons per high power field (h.p.f.). (A) Lesion volume, depicted in cubic mm, is similar among all injured groups. (B) CA3 hippocampal neuronal survival is worse in rats treated with fentanyl pre- and post-TBI, compared to any group pretreated with isoflurane (*P b 0.05 vs. iso:iso, iso:fent, iso:none). Post-traumatic treatment with isoflurane attenuates CA3 hippocampal neuronal death following fentanyl pretreatment (#P b 0.05 vs. fent:iso). (C) CA1 hippocampal neuronal death is worst in rats given fentanyl before and after TBI (†P b 0.05 vs. all other groups).
isoflurane, ketamine, morphine, pentobarbital, and propofol) in experimental TBI corroborates these findings. While we found that isoflurane-treated rats exhibited the best cognitive recovery and hippocampal survival, rats treated with fentanyl had outcomes similar to other anesthetic groups (Statler et al., 2000, 2001, 2003, 2006). Our findings suggest that, rather than being directly detrimental, fentanyl fails to afford neuroprotection (compared to isoflurane) in the controlled cortical impact model of experimental TBI. Further investigations, specifically focused on the interplay between fentanyl anes-
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thesia, secondary injury cascades, and neuronal excitation, are needed to further clarify the putative deleterious actions of fentanyl anesthesia in TBI. Recent work in cerebral ischemia has questioned the permanence of isoflurane neuroprotection (Elsersy et al., 2004; Kawaguchi et al., 2000, 2004). In these studies, early beneficial effects of isoflurane compared to fentanyl-treated (Kawaguchi et al., 2000, 2004) or awake (Elsersy et al., 2004) rats have dissipated by 2–3 weeks after the ischemic insult. In our study, isoflurane administration is associated with improved cognitive and histological outcomes 2–3 weeks after TBI, suggesting more long-lasting effects. Additional longitudinal studies are needed to more clearly delineate the long-term effects of isoflurane in experimental TBI. While the permanence of isoflurane neuroprotection remains unclear, given the widespread use of isoflurane anesthesia in experimental TBI, even transient benefits conferred by isoflurane may have important implications for study design and interpretation. It is interesting that post-traumatic isoflurane attenuated hippocampal neuronal death in rats treated with fentanyl just prior to TBI, yet failed to alter functional outcome. These findings may reflect a lack of correlation between the effects of isoflurane on functional and histopathological parameters. Other investigators have reported similar discrepancies between functional and histopathological outcomes after TBI (Clark et al., 2000; Varma et al., 2002; Whalen et al., 1999). Alternatively, isoflurane may promote subtle improvements in motor or cognitive performance. The functional and cognitive testing methods applied in our study may lack sufficient sensitivity to detect such subtle differences. Indeed, in clinical TBI studies, more intricate and complex tasks are often necessary to unmask post-traumatic functional deficits or improvements (Chapman et al., 2000). More complex testing batteries may similarly be necessary to reveal subtle differences between treatment groups in experimental TBI. Thus, the use of relatively simple (albeit standard) functional and cognitive outcome tests may be a limitation of our study. Hippocampal neuronal survival was assessed in our study using manual hippocampal neuronal counts in a single 10-μm coronal brain section taken 5-mm from the occiput and stained with cersyl violet. Cresyl violet does not stain neurons specifically. Surviving neurons were distinguished from glia based on cell morphology and size. We acknowledge that this assessment technique is not as rigorous as the use of neuronspecific staining techniques or classical stereological techniques. This is a limitation of our study. Further, more rigorous histopathological assessment is needed to better clarify the histological protection afforded by isoflurane in our study. The comparison of inhalational and intravenous anesthetic agents is another limitation of our study. It is difficult to compare anesthetic depth, as defined by the classic concept of minimal alveolar concentration (MAC), between these two techniques. We chose dosing regimens based on literature review (Miura et al., 1999; Soonthon-Brant et al., 1999) and pilot studies in our laboratory. The doses chosen are identical to those used in our prior investigations (Statler et al., 2000, 2003, 2006). Although no experimental group demonstrated signs of increased discomfort or stress, such as unexplained hypertension or increased hyperglycemia, equivalent anesthesia, as defined by the classic concept of MAC, may not have
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been achieved. Consequently, our results may reflect differences in the post-traumatic stress response, as well as differences in anesthetic actions. Nonetheless, we have compared dosing regimens of isoflurane and fentanyl that are commonly applied in experimental brain injury models and shown that rats treated with fentanyl prior to TBI showed worse outcomes. We have shown that isoflurane, a commonly used anesthetic in experimental TBI, confers beneficial effects even when administered at the lowest feasible level before TBI (i.e., when discontinued with recovery to tail pinch immediately before injury). In addition, administration of post-traumatic isoflurane produces benefits only in rats initially anesthetized with fentanyl and only on histopathology. Our results indicate that isoflurane exerts neuroprotective actions at or very near the time of TBI. Since it is inhumane to subject an awake animal to TBI, our findings have direct implications for study design and interpretation in experimental TBI. We are not advocating that use of isoflurane be abandoned; however, investigators using isoflurane must be cognizant of and give careful consideration to the beneficial effects of this anesthetic. Myriad factors dictate anesthetic choice in experimental TBI, and these clearly differ among studies. Investigators should weigh the effects of different anesthetic agents and tailor their choice accordingly. Although isoflurane is easy to use and commonly preferred, at times use of a less neuroprotective anesthetic, such as fentanyl, may be preferable.
4.
Experimental procedures
Adult, male (300–440 g), Sprague–Dawley rats were used in all experiments. The rats were allowed free access to food and water before and after surgery. The University of Pittsburgh Animal Care and Use Committee approved all studies. All surgical procedures were performed using aseptic technique. Rats were anesthetized with 4% isoflurane (Isoflo, Abbott Laboratories, North Chicago, IL) via nose cone and then endotracheally intubated with a 14-guage angiocatheter and mechanically ventilated. Anesthesia was maintained with 2–2.5% isoflurane for the duration of surgical preparation. Femoral vessels were cannulated for continuous arterial blood pressure monitoring, blood sampling, and medication administration. Pancuronium bromide (0.1 mg/kg/h, ElkinsSinn, Cherry Hill, NJ) was given for muscle relaxation. Core temperature was monitored via a rectal probe and controlled at 37 ± 0.5 °C. The rat was placed into a stereotaxic frame (David Kopf, Tujunga, CA) and a craniotomy (6 × 6mm) was performed over the left parietal cortex using a high-speed dental drill. The dura and bone flap were left in place until immediately before CCI. A temperature probe (2.28-mm outside diameter, Physitemp Corp., Clifton, NJ) was placed through a burr hole into the left frontal lobe, and brain temperature was controlled at 37 ± 0.5 °C. At the end of preparatory surgery, rats were randomized (n = 27 per group) to 30 min of either isoflurane or fentanyl before TBI. In the isoflurane group, isoflurane was continued at 1%. In the fentanyl group, isoflurane was stopped and fentanyl (50 mcg/ml, Elkins-Sinn, Cherry Hill, NJ) was
administered as a 10 mcg/kg iv bolus followed by a 50 mcg/kg/h iv infusion. In order to mimic clinical TBI, in which victims are not anesthetized at the time of injury, as close as humanely possible, either isoflurane or fentanyl was stopped at the end of the 30 min equilibration period. Rats were allowed to recover to tail pinch response and controlled cortical impact (6-mm tip, 4 m/s velocity, 1.8-mm depth of deformation) was performed immediately before rats had fully awakened from anesthesia. This anesthetic approach has been used in other TBI studies (Jenkins et al., 2002; Statler et al., 2006) and represents the lowest feasible, humane level of anesthesia at the time of TBI. Immediately after TBI, rats were randomized (n = 9 per group) to 1 h of isoflurane (1% by inhalation), fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h), or no additional anesthesia. (In order to facilitate humane closure of the craniotomy and skin incision, isoflurane 1% was administered for approximately 5 min post-TBI to rats in the no additional anesthesia groups.) The 1-h post-traumatic treatment period was chosen based on the initial posttraumatic period of maximal excitotoxicity (Palmer et al., 1993). Allowing rats to awaken immediately after TBI (i.e., administering no additional post-traumatic anesthesia) corresponds to the standard anesthetic approach used by several laboratories (Hallam et al., 2004; Kozlowski et al., 2004), including ours (Kochanek et al., 1995). This study design created six anesthetic groups (n = 9 per group), based on pre:post-TBI anesthesia: isoflurane:isoflurane, isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, and fentanyl:none. As an additional control, shams underwent craniotomy using isoflurane anesthesia with recovery to tail pinch, but no TBI or additional anesthesia. At the end of the 1-h treatment period, rats were allowed to awaken and recovered to extubation. Mean arterial blood pressure (MAP) and rectal and brain temperatures were monitored continuously during the 1h treatment period. Blood glucose levels, arterial blood gas samples, and hematocrit were assessed every 15 min. PaCO2 was controlled at 35–45 mm Hg, and PaO2 was greater than 100 mm Hg throughout the experiment in all preparations. Motor function was assessed by beam walking and beam balance tasks on days 1–5 after injury by an observer masked to experimental group. Cognitive outcome was assessed using a submerged-platform paradigm of the Morris water maze (MWM) on days 14–18 after injury by an observer masked to experimental group. On postinjury day 21, rats were reanesthetized with isoflurane 4% and perfused with heparinized saline followed by 4% paraformaldehyde. Brains were removed, postfixed, and cryoprotected. Serial coronal sections (10-μm) were made at 1-mm intervals throughout the brain, mounted on slides, and stained with cresyl violet. Lesion volume was assessed by an observer masked to experimental group, using an image analysis system (MCID, Imaging Research, St. Catherine's, Ontario, Canada). Surviving hippocampal neurons, identified by morphology and size, were counted by an observer masked to experimental group, under ×400 magnification in the anatomic CA1 and CA3
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hippocampal regions in a single 10-μm coronal section taken 5-mm from the occiput.
4.1.
Statistical analysis
All statistical analyses were performed using either SPSS, version 11 for MAC OS X or SAS, version 9.1 for Windows. Arterial blood gas values, blood glucose levels, and MAP were compared using two-way analysis of variance for repeated measures. Beam balance and beam walking results and MWM latencies were compared using a linear mixed model with experimental group and time-specified as categorical main effects. An unstructured covariance matrix was used to model the association between measures for each subject over time. The duration of preparatory surgery, lesion volume, and hippocampal neuronal survival were compared using one-way analysis of variance. Tukey's test was used in all post hoc comparisons, where appropriate. A P b 0.05 was considered significant. Data are presented as mean ± SEM.
Acknowledgments We thank the U.S. Army (DMAD 17-91-7009), the National Institutes of Health (NS30318 and T32-HD40686), and the Laerdal Foundation for Acute Medicine for generous support of this project. We also thank Marci Provins for assistance with preparation of the manuscript.
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