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Morphological maladaptations in sympathetic preganglionic neurons following an experimental high-thoracic spinal cord injury
Experimental Neurology 327 (2020) 113235
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Research Paper
Morphological maladaptations in sympathetic preganglionic neurons following an experimental high-thoracic spinal cord injury Rahul Sachdevaa,b, Gillian Huttona,b, Arshdeep S. Marwahaa,b, Andrei V. Krassioukova,b,c,
T ⁎
a
International Collaboration on Repair Discoveries (ICORD), University of British Columbia, Vancouver, Canada Department of Medicine, Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, Canada c GF Strong Rehabilitation Center, Vancouver Coastal Health, Vancouver, Canada b
A R T I C LE I N FO
A B S T R A C T
Keywords: Spinal cord injury Sympathetic preganglionic neurons Cardiovascular function Morphometry
Spinal cord injury (SCI) disrupts the supraspinal vasomotor pathways to sympathetic preganglionic neurons (SPNs) leading to impaired blood pressure (BP) control that often results in episodes of autonomic dysreflexia and orthostatic hypotension. The physiological cardiovascular consequences of SCI are largely attributed to the plastic changes in spinal SPNs induced by their partial deafferentation. While multiple studies have investigated the morphological changes in SPNs following SCI with contrasting reports. Here we investigated the morphological changes in SPNs rostral and caudal to a high thoracic (T3) SCI at 1-, 4- and 8-weeks post injury. SPNs were identified using Nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH- diaphorase) staining and were quantified for soma size and various dendritic measurements. We show that rostral to the lesion, soma size was increased at 1 week along with increased dendritic arbor. The total dendritic length was also increased at chronic stage (8 weeks post SCI). Caudal to the lesion, the soma size or dendritic lengths did not change with SCI. However, dendritic branching was enhanced within a week post SCI and remained elevated throughout the chronic stages. These findings demonstrate that SPNs undergo significant structural changes form sub-acute to chronic stages post-SCI that likely determines their functional consequences. These changes are discussed in context of physiological cardiovascular outcomes post-SCI.
1. Introduction In addition to sensory and motor impairments, SCI results in a myriad of debilitating autonomic dysfunctions that severely impact the survival and quality of life post injury (Hou and Rabchevsky, 2014; Krassioukov, 2009). With remarkable strides being made in acute care and management of other secondary conditions such as urinary tract infections, septicemia, renal dysfunction and pneumonia, cardiovascular dysfunction emerges as one of the leading causes of morbidity and mortality following SCI (Cragg et al., 2013; Garshick et al., 2005). Understandably, cardiovascular health is among the highest priorities for recovery in the individuals with SCI (Anderson, 2004; Hammell, 2010). In addition to having a consistently low resting blood pressure (BP), individuals with tetraplegia or high paraplegia (SCI above sixth thoracic segment; T6) frequently undergo dramatic BP fluctuations (Sachdeva et al., 2019). Within the same individual with SCI, while an orthostatic challenge (e.g., assuming an upright posture) can cause the systolic BP to drop down to 50 mmHg or lower (orthostatic
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hypotension), a noxious or non-noxious stimulus originating below the spinal cord lesion (e.g., distended bladder) can trigger a sudden rise in BP reaching up to 300 mmHg (autonomic dysreflexia). These hypotensive and hypertensive crises are the result of either insufficient or excessive vasoconstriction respectively, primarily as a result of maladaptive changes in spinal SPNs below the lesion (Krassioukov, 2009). SPNs, located in four regions of spinal cord (the intermediolateral cell column, the intercalated nucleus, central autonomic nucleus and the lateral funiculus) between the T1 and L2 segments, determine the final sympathetic outflow from the central nervous system (CNS), regulating various autonomic functions including the BP (Llewellyn-Smith, 2009). Although cardiovascular dysfunction has been reported with SCI as low as T10 spinal segment (Gimovsky et al., 1985), it is most frequently observed in individuals with lesions above T6 segment, below which lie the SPNs responsible for modulating the highly compliant splanchnic and lower extremity blood vessels (i.e. the primary capacitance vascular bed). Despite complex pathophysiological alterations in central as well as peripheral components of the autonomic nervous system, cardiovascular dysfunction following SCI is largely attributed to the
Corresponding author at: ICORD-BSCC, 818 West 10th Avenue, Vancouver, BC V5Z 1M9, Canada. E-mail address: [email protected] (A.V. Krassioukov).
https://doi.org/10.1016/j.expneurol.2020.113235 Received 10 July 2019; Received in revised form 15 January 2020; Accepted 6 February 2020 Available online 07 February 2020 0014-4886/ © 2020 Elsevier Inc. All rights reserved.
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2.2. Tissue processing
changes resulting from decentralization of SPNs (Biering-Sorensen et al., 2017). Within the past two decades, a number of research groups, including ours have utilized human and rodent spinal cords to provide crucial insights into structural and functional maladaptations of SPNs, leading to abnormal sympathetic control of arterial pressure following SCI (Kalincik et al., 2010; Krassioukov et al., 1999; Krassioukov and Weaver, 1995, 1996; Lujan et al., 2010). However, a considerable lack of agreement exists among the specific findings reported in these studies e.g. temporal changes in soma size as well as dendritic arbor rostral and caudal to the injury site. A clear understanding of the time course of morphological changes in SPNs is important in unraveling of the mechanisms underlying autonomic cardiovascular dysfunction following SCI. In the present study we sought to address some of the discrepancies put forward across previous studies investigating the morphology of SPNs following SCI. Using a T3 complete transection model of SCI, we investigated the morphometric changes in a large population of SPNs in the spinal cord rostral and caudal to injury, by staining for NADPHdiaphorase. The results obtained in this study are discussed in context of previous literature.
At each experimental time point, the animals were euthanized with an overdose of chloral hydrate (Trichloroacetaldehyde monohydrate, 1 g/Kg, i.p.) and perfused transcardially with 150 ml of 0.1 M Phosphate buffer (pH 7.4), followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) (Krassioukov and Weaver, 1996). The spinal cord was removed and post-fixed in 4% paraformaldehyde for 12 h and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer for a minimum of 72 h (Sachdeva et al., 2016a). Thoracic spinal cord segments from C8 to T7 were embedded in Cryomatrix®, frozen in liquid nitrogen and stored at −80 °C prior to sectioning. The spinal cord segments were cut on a Cryostat® at 30 μm thick horizontal longitudinal sections, followed by mounting on Superfrost® Plus glass slides and stored at −20 °C. 2.3. Histochemical identification of SPNs using NADPH-diaphorase NADPH-diaphorase is a nitric oxide synthase enzyme constitutively expressed by spinal SPNs. This enzyme is capable of transferring hydrogen from NADPH to a soluble tetrazolium salt, converting it into insoluble, stable and distinctly visible formazan (Kluchova et al., 2001; Krassioukov and Weaver, 1996). Briefly, a hydrophobic barrier was made on the glass slide around the tissue using rubber cement (Elmer's®). Tissue sections were washed in 1 × phosphate buffer saline (PBS) solution twice for 10 min on a bench top orbital shaker. The tissue was incubated at 37 °C for 15 h in a solution of 5% nitro-blue tetrazolium chloride (NBT; ThermoFisher Scientific®), 2% NADPH (Sigma-Aldrich®) in 0.2% Triton-1 × PBS solution. Following incubation, rubber cement was removed and the slides were washed three times for 10 min in 1 × PBS solution (pH 7.4), and once in 100% alcohol for 15 min. The slides were air-dried for 2 h and cover-slipped using Cytoseal®. Sections were imaged under at 40 × magnification with an Aperio CS2 slide scanner.
2. Materials and methods Animal surgery and post-surgical care protocols were approved by the University of British Columbia Animal Care Committee and were performed in strict compliance with the policies established by the Canadian Council on Animal Care (institutional approval certificate number: A18–0183).
2.1. Spinal cord injury Twenty-four adult (~300 g), male Wistar rats (Harlan Laboratories, Indianapolis, IN) were divided across four groups (n = 6 each). Three groups received SCI and were euthanized at 1-, 4- and 8-weeks post injury. A fourth group received no SCI and served as an uninjured control. Surgical procedures and post-operative care were performed as published previously (Phillips et al., 2018; Ramsey et al., 2010). Starting three days prior to surgery, a subcutaneous injection of prophylactic enrofloxacin (Baytril; 10 mg/kg, Associated Veterinary Purchasing; AVP, Langley, Canada) was administered daily. On the day of the surgery, animals were anaesthetized with isoflurane using a 5% induction dose and maintained under surgical plane at 1.5–2%. Subcutaneous injections of enrofloxacin (10 mg/kg), buprenorphine (Temgesic®; 0.02 mg/kg, McGill University), and ketoprofen (Anafen®, 5 mg/kg, AVP) were administered pre-operatively and continued for 3 days post-surgery. Following a dorsal midline skin incision and blunt dissection of superficial muscles overlying C8 to T4, a laminectomy was performed at T2 vertebra to expose the T3 spinal cord segment. Dura mater was incised using a 30G needle and a complete transection was made using micro-scissors and gentle vacuum aspiration (Sachdeva et al., 2016b). Lesion completeness was confirmed using visual inspection and a pledget of Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) was placed between the stumps to achieve hemostasis. Dura was sutured close using 9–0 Prolene sutures (Sachdeva et al., 2016a). The muscle and skin were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively. Following surgery, animals received 5 ml subcutaneous injections of warm Lactated Ringer's solution (Baxter Corp., Canada) and allowed to recover in a temperature-controlled environment (Animal Intensive Care Unit, Los Angeles, CA, USA) before returning to home cages. Urinary bladders were manually expressed two to three times daily until spontaneous voiding returned (~ 10 days post injury).
2.4. Morphometric analysis and quantification Large scans of longitudinal spinal cord sections were visualized using the ImageScope software (Leica Biosystems). Individual neuron snapshots were randomly taken from two segments rostral and caudal to the lesion. Only neurons that met the criteria of having non-overlapping dendrites, distinguishable soma and uniform staining were included in quantification. A minimum of 20 neurons were analyzed per animal from at least 3 non-consecutive sections. Morphometric analysis was performed using the Fiji (Fiji Is Just ImageJ; https://fiji.sc/) application and was done blinded to the animal group and the neuron position in relation to the injury. All neuronal snapshots were blinded by a researcher unrelated to the study. Images were calibrated to scale and converted to 8-bit image type before analysis. The ImageJ freehand selection tool was used to trace the soma perimeter and data for soma area were calculated. Neurites were traced using the Simple Neurite Tracer Segmentation plugin (https://imagej. net/Simple_Neurite_Tracer) and analyzed using the Hessian-based analysis tools. The primary number of dendrites, total dendritic length summed from all dendrites and the maximum individual dendrite length per neuron were recorded. The Sholl analysis plugin (https:// imagej.net/Sholl_Analysis) was used to analyze all paths. Concentric rings with a radius step size of 10 μm were superimposed around the neuron soma center. Number of dendritic intersections at each concentric ring was summed and recorded as an indirect measure of dendritic arborisation (Lujan et al., 2010). 2.5. Statistical analyses GraphPad Prism software was used for statistical analyses. Two-way ANOVA (group vs. rostral/caudal location) was used to compare SPN 2
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Fig. 1. Identification of SPNs in spinal cord. (A) A longitudinal section of injured spinal cord stained for NADPH-diaphorase. Rectangular boxes indicate distinct regions containing SPNs magnified in (B) intermediolateral column, IML, (C) central autonomic area, CAA, and (D) intercalated nucleus, ICN. * indicates the lesion at T3.
Fig. 2. Quantification of soma size. (A) photomicrograph showing three NADPH-diaphorase-stained neurons showing cross sectional area of the soma highlighted using the freehand selection tool on ImageJ. (B) comparison of soma area in spinal SPNs rostral and caudal to injury showing a significant increase in soma size rostral to lesion at 1-week post-SCI. * indicates statistical significance, p < .05.
3.2. Soma size
soma size area, number of primary dendrites per neuron, maximum dendritic length per neuron and total dendritic length per neuron. For Sholl analysis results, two-way ANOVA (group vs. intersections) was performed to compare the number of dendritic intersections at consecutive 10 μm interval concentric rings. For all analyses, HolmŠídák's post hoc test was used for multiple comparisons. All data are reported as mean ± standard deviation (SD). In all analyses, data were considered statistically significance at p < .05.
Soma size was calculated as the 2-dimensional area by manually tracing the perimeter in ImageJ (Fig. 2A). Soma area was elevated at one-week post injury compared to uninjured controls in the neurons rostral to injury (Fig. 2B, Table 1). No differences were seen at other location or other timepoints. 3.3. Number of primary dendrites, maximum dendritic length, total dendritic length
3. Results
Following a semi-automated tracing of the dendrites, total dendritic length was summed from all dendrites and the maximum individual dendrite length per neuron were recorded (Fig. 3A). The number of primary dendrites were also counted manually for each neuron. Total dendritic length (Fig. 3B, Table 1) in the neurons rostral to the injury did not change at 1- and 4-weeks post SCI but was found to be significantly longer at 8-weeks post SCI. No effect of SCI was seen on the total dendritic length of the neurons below the lesion at any time points. Maximum individual dendritic length per neuron (Fig. 3C, Table 1) as well as the number of primary dendrites per neuron (Fig. 3D, Table 1) remained unchanged above and below the lesion following SCI.
3.1. Identification of SPNs Using the NADPH-diaphorase staining, spinal SPNs were identified in four distinct regions of a spinal cord section (see Fig. 1 for example). Majority of the SPNs were localized in close proximity to each other within the intermediolateral (IML) column at the junction of white and grey matter (Fig. 1B). Other spinal regions containing SPNs included the central autonomic area (CAA) near central canal (Fig. 1C), the intercalated nucleus (ICN; region between IML and CAA, Fig. 1D). A small proportion of neuronal cell bodies were also observed in the dorsolateral funiculus (not shown). No differences were observed between the right and left side of the spinal cord in terms of distribution or structure of neurons. Following the image acquisition, neurons within two segments rostral and caudal to injury (and corresponding segments from uninjured spinal cords) were randomly captured for analyses, while excluding the neurons with incomplete staining, truncated dendrites, excessive overlap with adjacent neurons. The neurons included in the quantification were free of bias towards the distinct regions i.e. IML, CAA and ICN.
3.4. Dendritic branching pattern: sholl analysis In order to assess the branching pattern of SPN dendrites, Sholl analysis (originally referred to as concentric shell method) was employed (Sholl, 1953). This quantitative analysis method allows the investigation of dendritic arborization using concentric circles as 3
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8 weeks
356.94 ± 97.00 420.13 ± 87.38 268.04 ± 62.71 2.89 ± 0.10
4 weeks
428.82 ± 119.88 323.27 ± 59.31 199.13 ± 22.58 2.53 ± 0.37
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coordinates of reference originating from the cell body. The extent of dendritic arbor indirectly represents the available postsynaptic space on the SPNs. For the present study, the concentric circles were calibrated at 10 μm step size and the number of dendritic intersections with circle at each step size were quantified (Fig. 4A). Rostral to the lesion, SCI led to an increase in the number of intersections (i.e. dendritic branching) at 1-week post injury and this effect was not significant at the later timepoints (Fig. 4B). More robust effect of SCI on dendritic branching was seen caudal to injury where SCI led to a significant increase in dendritic arbor at all three timepoints, suggesting a consistent increase in available postsynaptic space (Fig. 4C).
475.55 ± 70.33 358.82 ± 71.73 217.89 ± 47.91 2.58 ± 0.47 347.471 ± 127.5 446.19 ± 133.42 256.59 ± 36.85 2.52 ± 0.20 464.93 ± 65.33 640.52b ± 223.03 335.32 ± 167.92 3.11 ± 0.37
In order to understand the region-specific effect on SPN subpopulations, the morphometric characteristics of SPNs were further analyzed based on their localization in autonomic nuclei (Fig. 5). Within ICN, SPNs showed significant increase in soma size rostral to the lesion as early as 7 days post injury, which remained elevated for the remaining timepoints. Caudal to the lesion, ICN neurons showed an SCIdependent increase in soma size at 4 weeks post injury (Fig. 5A). A similar increase in soma size were also seen in SPNs within CAA caudal to the lesion at 7 days post injury (Fig. 5B). The maximum dendritic length of CAA neurons rostral to the injury was increased at 8 weeks post injury (Fig. 5C). No statistically significant differences were seen in the SPNs within IML (data not shown). 4. Discussion Using a high thoracic (T3) SCI, this study demonstrates the time course of morphological changes in SPNs rostral and caudal to injury. T3 complete SCI was utilized because this injury model decentralizes the majority of SPNs from the supraspinal cardiovascular control, while sparing two segments (T1 and T2) that provide sufficient SPNs to be studied as rostral, supraspinally connected controls. This injury model has been extensively characterized by our group and others in terms of cardiovascular dysfunction e.g. autonomic dysreflexia (Krassioukov et al., 2002; West et al., 2015b), cardiac structure and functional impairments (Poormasjedi-Meibod et al., 2019; Squair et al., 2018a), cerebrovascular (Phillips et al., 2016), gastrointestinal (Frias et al., 2018) as well as cardiometabolic dysfunctions (Inskip et al., 2010). Furthermore, using this model, we have also shown many promising treatment strategies resulting in significant cardiovascular recovery (Sachdeva, 2017; Squair et al., 2018b; West et al., 2015a). In this study, we observed that a high thoracic SCI leads to significant structural maladaptations in SPNs responsible for autonomic (e.g. cardiovascular) function. Rostral to the injury, SCI resulted in an early increase in soma size (at 1 week) that persisted for longer timepoints (i.e. 4 and 8 weeks timepoints were not different from 1 week). Dendrites on the other hand, did not change initially post injury but the total dendritic length was significantly greater at 8 weeks post SCI. Caudal to the injury, although no significant differences were seen in soma size or dendritic lengths, the dendrites showed significantly more branching compared to uninjured controls within a week post SCI and lasted for later time points. Interestingly, we also report region-specific responses to SCI by specific SPN subpopulations. Given the previously established location-specific neurochemical differences within SPNs (Hinrichs and Llewellyn-Smith, 2009), these results support the idea of structural and functional diversity observed within autonomic neurons (Llewellyn-Smith, 2009). This may have important implications in future research targeting the SPNs by therapeutic approaches for functional recovery (Kalincik et al., 2010). In context of previous pertinent literature, some similarities and differences exist. Our results are partially in agreement with those of Kalincik et al., which showed that both rostral and caudal to the SCI,
325.31 ± 130.07 364.21 ± 90.16 231.52 ± 41.45 2.69 ± 0.85 Soma size (μm ) Total dendritic length (μm) Maximum dendritic length (μm) Number of primary dendrites
Each cell represents mean ± SD for the group. a Indicates significant difference from the uninjured group. b Indicates significant difference from uninjured, 1 week and 4 weeks.
524.82 ± 121.42 379.90 ± 61.10 225.78 ± 34.06 2.85 ± 0.51
465.09 ± 116.57 330.37 ± 58.85 201.50 ± 50.35 2.69 ± 0.10
Uninjured 8 weeks 4 weeks 1 week Uninjured
2
Rostral to injury Analysis
Table 1 Morphometric measurements of SPNs following SCI.
a
Caudal to injury
1 week
3.5. Region-specific morphometric analyses
4
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Fig. 3. Quantification of number and length of dendrites. (A) photomicrograph showing dendrites traced using semi-automated Simple Neurite Tracer (ImageJ). (B) total dendritic length was significantly increased at 8-weeks post injury. No effect of SCI was seen on the maximum dendritic length (C) or the number of primary dendrites (D). * indicates statistical significance, p < .05.
Fig. 4. Quantification of dendritic branching. (A) An example image of a neuron analyzed via Sholl analysis, with 10 μm step size concentric rings superimposed around the soma (not shown to scale). Intersections within each concentric ring were summed as an indirect measurement of dendritic branching. (B) and (C) show significant increase in dendritic arborization of SPNs rostral and caudal to the lesion respectively. *, φ, and Ψ indicate statistical significance (p < .05) between uninjured control and 1-week, 4-weeks and 8-weeks respectively.
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Fig. 5. Region-specific morphometric analyses. (A) comparison of soma area within ICN SPNs rostral and caudal to injury showing a significant increase in soma size rostral to lesion at all time points post-SCI and at 4 weeks post SCI caudal to the lesion. (B) Within CAA, the soma area was increased caudal to the lesion at 7 days post injury. (C) Within CAA, the maximum dendritic length was increased rostral to the lesion at 8 weeks post injury. * indicates statistical significance, p < .05.
vasoconstriction in order to restore BP, despite a normal feedback via the arterial baroreflex system. Concomitantly on the other hand, the decentralized SPNs remain prone to be influenced by inputs arising from below the lesion. Consistent with previous studies, we show that this possibility may be further enhanced by increased dendritic branching of SPNs following SCI, putatively resulting in enhanced postsynaptic space. It is possible that increased dendritic arbor is accompanied by higher metabolic demand leading to increased soma size. Even though autonomic dysreflexia has also been reported in acute stages (Krassioukov et al., 2003), it is more commonly present in subacute and chronic stages- consistent with the aberrant sprouting of primary sensory afferents (Krenz and Weaver, 1998), suggesting that stimuli arising from primary sensory afferents below the lesion result in excitation of the SPNs leading to autonomic dysreflexia, most likely via multisynaptic long propriospinal pathways (Schramm, 2006).
soma size is increased, whereas dendritic length and number of primary dendrites are transiently increased (Kalincik et al., 2010). While we observed similar somal and dendritic effects rostral to injury, we did not see increased dendritic length caudal to injury. In our experiments, dendritic branching was significantly enhanced both rostral and caudal to injury but this parameter was unfortunately not investigated by Kalincik et al. A similar study by Lujan et al. also reported an SCI-dependent increase in soma size, dendritic length and branching rostral to injury in cardiac SPNs (Lujan et al., 2010). Our observations in SPNs rostral to SCI agree with this study despite the fact that this study analyzed a subpopulation of SPNs that project to stellate ganglia, in contrast to a more global population identified by NADPH-diaphorase staining. This study, however did not report the structural analyses in SPNs caudal to the injury site and hence that region cannot be compared. Finally, a discrepancy exists regarding whether there is an initial (1 week post-SCI) reduction in SPN soma size caudal to injury as reported previously (Krassioukov and Weaver, 1996). In contrast to this previous report, the present study agrees with the abovementioned studies by Kalincik et al. and Lujan et al. However, the initial discrepancy can be explained in at least two ways. Firstly, the previous study analyzed only a specific subpopulation of SPNs that project to adrenal medulla. It is possible that those SPNs behave differently and may not be an ideal representation of the total population of SPNs. This is partly supported by the observations that SPNs within different regions of spinal cord grey matter have specific structural and functional identities (Llewellyn-Smith, 2011; Wu et al., 2012). This has also been confirmed in the region-specific analyses in present study. Secondly, the reduction in soma size caudal to injury reported by Krassioukov and Weaver was shown in comparison to the SPNs rostral to injury, not to the SPNs in uninjured spinal cords (Krassioukov and Weaver, 1996). Therefore, this observed difference was likely due to the fact that the SPNs rostral to injury show a significant increase in soma size.
5. Summary The mechanisms underpinning cardiovascular consequences of SCI are enigmatic as the affected individuals can experience two opposite BP extremes within hours. Understanding the SCI-induced changes in key players involved in cardiovascular control is important in developing prevention/treatment strategies. We present and discuss temporal changes in SPNs following an experimental SCI that results in cardiovascular dysfunction and attempt to address some discrepancies across previous literature. A limitation of the present study is that although two-dimensional assessment of neuronal morphology is a standard procedure in the field, it does not account for dendritic arborization in z-dimension. Another inherent methodological limitation is that a large population of neurons, especially in the IML did not meet the inclusion criteria for analysis and were excluded. While orthostatic hypotension and autonomic dysreflexia are partly explained by SCIrelated changes in SPNs, it is important to mention that this discussion does not consider the numerous peripheral components contributing to cardiovascular dysfunction such as impaired renin-angiotensin system, cardiac deconditioning, reduction in plasma volume, fibrotic as well as hyper-responsiveness vasculature etc. A better understanding of these factors and their interplay is crucial towards a better understanding of cardiovascular impairments following SCI.
4.1. Implications for cardiovascular dysfunction Sympathetic activity and in turn, cardiovascular dysfunction following SCI is somewhat perplexing since it ranges from abnormally low to abnormally high BP within the same individual. SPNs being the final neurons determining the CNS sympathetic output are often regarded as key regulators of BP. It has been long known that SPNs do not exhibit intrinsic pacemaker potentials (Laskey and Polosa, 1988). Hence their activity in an intact nervous system is largely determined by spinal and supraspinal synaptic inputs (Guyenet, 2006). With the SCI-induced loss of supraspinal sympatho-modulatory input, the efferent sympathetic activity of SPNs is diminished. With significantly diminished tonic and reflex autonomic activity, individuals with a high-thoracic or cervical SCI experience a low resting BP as well as a substantial decline in BP while assuming an upright position (i.e. orthostatic hypotension). This is a consequence of the inability of supraspinal pathways to trigger
Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements The present study is supported by funds from Canadian Institutes of Health Research (CIHR) and Wings for Life Foundation. Dr. Krassioukov's laboratory is also supported by funds from the Heart and Stroke Foundation, Canadian Foundation for Innovation, BC Knowledge 6
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Development Fund, Craig H. Neilsen Foundation and Seed grants from International Collaboration on Repair Discoveries (ICORD), supported by the Rick Hansen Foundation. Dr. Sachdeva is supported by Postdoctoral Fellowships from the Craig H. Neilsen Foundation, CIHR, Michael Smith Foundation for Health Research and University of British Columbia (Bluma Tischler Postdoctoral Fellowship). Mr. Marwaha's funding support is provided in part by the University of British Columbia Faculty of Medicine Summer Student Research Program. We would also like to thank Dr. David Granville and Mr. Yuan Jiang (ICORD) for their assistance with imaging experiments.
somatic and visceral stimuli after chronic spinal injury. J. Neurotrauma 19, 1521–1529. Krassioukov, A.V., Furlan, J.C., Fehlings, M.G., 2003. Autonomic dysreflexia in acute spinal cord injury: an under-recognized clinical entity. J. Neurotrauma 20, 707–716. Krenz, N.R., Weaver, L.C., 1998. Sprouting of primary afferent fibers after spinal cord transection in the rat. Neuroscience 85, 443–458. Laskey, W., Polosa, C., 1988. Characteristics of the sympathetic preganglionic neuron and its synaptic input. Prog. Neurobiol. 31, 47–84. Llewellyn-Smith, I.J., 2009. Anatomy of synaptic circuits controlling the activity of sympathetic preganglionic neurons. J. Chem. Neuroanat. 38, 231–239. Llewellyn-Smith, I.J., 2011. Sympathetic Preganglionic Neurons. In: Llewellyn-Smith, Ida J., Verberne, A.J.M. (Eds.), Central Regulation of Autonomic Functions, 2nd ed. Oxford University Press, pp. 98–119. Lujan, H.L., Palani, G., DiCarlo, S.E., 2010. Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization. Am. J. Phys. Regul. Integr. Comp. Phys. 299, R985–R995. Phillips, A.A., Matin, N., Frias, B., Zheng, M.M., Jia, M., West, C., Dorrance, A.M., Laher, I., Krassioukov, A.V., 2016. Rigid and remodelled: cerebrovascular structure and function after experimental high-thoracic spinal cord transection. J. Physiol. 594, 1677–1688. Phillips, A.A., Matin, N., Jia, M., Squair, J.W., Monga, A., Zheng, M.M.Z., Sachdeva, R., Yung, A., Hocaloski, S., Elliott, S., Kozlowski, P., Dorrance, A.M., Laher, I., Ainslie, P.N., Krassioukov, A.V., 2018. Transient hypertension after spinal cord injury leads to cerebrovascular endothelial dysfunction and fibrosis. J. Neurotrauma 35, 573–581. Poormasjedi-Meibod, M.S., Mansouri, M., Fossey, M., Squair, J.W., Liu, J., McNeill, J.H., West, C.R., 2019. Experimental spinal cord injury causes left-ventricular atrophy and is associated with an upregulation of proteolytic pathways. J. Neurotrauma 36, 950–961. Ramsey, J.B., Ramer, L.M., Inskip, J.A., Alan, N., Ramer, M.S., Krassioukov, A.V., 2010. Care of rats with complete high-thoracic spinal cord injury. J. Neurotrauma 27, 1709–1722. Sachdeva, R.G.R., Jia, M., Monga, A., Ramer, M., Krassioukov, A.V., 2017. A triple combination approach involving nerve transplantation, glial scar digestion and passive exercise promotes cardiovascular recovery after spinal cord injury. FASEB J. 31 1077.1077–1077.1077. Sachdeva, R., Farrell, K., McMullen, M.K., Twiss, J.L., Houle, J.D., 2016a. Dynamic changes in local protein synthetic machinery in regenerating central nervous system axons after spinal cord injury. Neural Plast. 2016, 4087254. Sachdeva, R., Theisen, C.C., Ninan, V., Twiss, J.L., Houle, J.D., 2016b. Exercise dependent increase in axon regeneration into peripheral nerve grafts by propriospinal but not sensory neurons after spinal cord injury is associated with modulation of regeneration-associated genes. Exp. Neurol. 276, 72–82. Sachdeva, R., Nightingale, T.E., Krassioukov, A.V., 2019. The blood pressure pendulum following spinal cord injury: implications for vascular cognitive impairment. Int. J. Mol. Sci. 20. Schramm, L.P., Polosa, L.C.W.A.C., 2006. Spinal sympathetic interneurons: Their identification and roles after spinal cord injury. In: Progress in Brain Research. Elsevier, pp. 27–37. Sholl, D.A., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87, 387–406. Squair, J.W., Liu, J., Tetzlaff, W., Krassioukov, A.V., West, C.R., 2018a. Spinal cord injuryinduced cardiomyocyte atrophy and impaired cardiac function are severity dependent. Exp. Physiol. 103, 179–189. Squair, J.W., Ruiz, I., Phillips, A.A., Zheng, M.M.Z., Sarafis, Z.K., Sachdeva, R., Gopaul, R., Liu, J., Tetzlaff, W., West, C.R., Krassioukov, A.V., 2018b. Minocycline reduces the severity of autonomic Dysreflexia after experimental spinal cord injury. J. Neurotrauma 2861–2871. West, C.R., Crawford, M.A., Laher, I., Ramer, M.S., Krassioukov, A.V., 2015a. Passive hind-limb cycling reduces the severity of autonomic Dysreflexia after experimental spinal cord injury. Neurorehabil. Neural Repair 317–327. West, C.R., Popok, D., Crawford, M.A., Krassioukov, A.V., 2015b. Characterizing the temporal development of cardiovascular dysfunction in response to spinal cord injury. J. Neurotrauma 32, 922–930. Wu, L., Chang, H.H., Havton, L.A., 2012. The soma and proximal dendrites of sympathetic preganglionic neurons innervating the major pelvic ganglion in female rats receive predominantly inhibitory inputs. Neuroscience 217, 32–45.
References Anderson, K.D., 2004. Targeting recovery: priorities of the spinal cord-injured population. J. Neurotrauma 21, 1371–1383. Biering-Sorensen, F., Biering-Sorensen, T., Liu, N., Malmqvist, L., Wecht, J.M., Krassioukov, A., 2017. Alterations in cardiac autonomic control in spinal cord injury. Auton. Neurosci. 4–18. Cragg, J.J., Noonan, V.K., Krassioukov, A., Borisoff, J., 2013. Cardiovascular disease and spinal cord injury: results from a national population health survey. Neurology 81, 723–728. Frias, B., Phillips, A.A., Squair, J.W., Lee, A.H.X., Laher, I., Krassioukov, A.V., 2018. Reduced colonic smooth muscle cholinergic responsiveness is associated with impaired bowel motility after chronic experimental high-level spinal cord injury. Auton. Neurosci. 33–38. Garshick, E., Kelley, A., Cohen, S.A., Garrison, A., Tun, C.G., Gagnon, D., Brown, R., 2005. A prospective assessment of mortality in chronic spinal cord injury. Spinal Cord 43, 408–416. Gimovsky, M.L., Ojeda, A., Ozaki, R., Zerne, S., 1985. Management of autonomic hyperreflexia associated with a low thoracic spinal cord lesion. Am. J. Obstet. Gynecol. 153, 223–224. Guyenet, P.G., 2006. The sympathetic control of blood pressure. Nat. Rev. Neurosci. 7, 335–346. Hammell, K.R., 2010. Spinal cord injury rehabilitation research: patient priorities, current deficiencies and potential directions. Disabil. Rehabil. 32, 1209–1218. Hinrichs, J.M., Llewellyn-Smith, I.J., 2009. Variability in the occurrence of nitric oxide synthase immunoreactivity in different populations of rat sympathetic preganglionic neurons. J. Comp. Neurol. 514, 492–506. Hou, S., Rabchevsky, A.G., 2014. Autonomic consequences of spinal cord injury. Comp. Physiol. 4, 1419–1453. Inskip, J., Plunet, W., Ramer, L., Ramsey, J.B., Yung, A., Kozlowski, P., Ramer, M., Krassioukov, A., 2010. Cardiometabolic risk factors in experimental spinal cord injury. J. Neurotrauma 27, 275–285. Kalincik, T., Jozefcikova, K., Sutharsan, R., Mackay-Sim, A., Carrive, P., Waite, P.M., 2010. Selected changes in spinal cord morphology after T4 transection and olfactory ensheathing cell transplantation. Auton. Neurosci. 158, 31–38. Kluchova, D., Rybarova, S., Miklosova, M., Lovasova, K., Schmidtova, K., Dorko, F., 2001. Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study. Eur. J. Histochem. 45, 239–248. Krassioukov, A., 2009. Autonomic function following cervical spinal cord injury. Respir. Physiol. Neurobiol. 169, 157–164. Krassioukov, A.V., Weaver, L.C., 1995. Reflex and morphological changes in spinal preganglionic neurons after cord injury in rats. Clin. Exp. Hyperten. (New York) 17, 361–373. Krassioukov, A.V., Weaver, L.C., 1996. Morphological changes in sympathetic preganglionic neurons after spinal cord injury in rats. Neuroscience 70, 211–225. Krassioukov, A.V., Bunge, R.P., Pucket, W.R., Bygrave, M.A., 1999. The changes in human spinal sympathetic preganglionic neurons after spinal cord injury. Spinal Cord 37, 6–13. Krassioukov, A.V., Johns, D.G., Schramm, L.P., 2002. Sensitivity of sympathetically correlated spinal interneurons, renal sympathetic nerve activity, and arterial pressure to
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Research Paper
Morphological maladaptations in sympathetic preganglionic neurons following an experimental high-thoracic spinal cord injury Rahul Sachdevaa,b, Gillian Huttona,b, Arshdeep S. Marwahaa,b, Andrei V. Krassioukova,b,c,
T ⁎
a
International Collaboration on Repair Discoveries (ICORD), University of British Columbia, Vancouver, Canada Department of Medicine, Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, Canada c GF Strong Rehabilitation Center, Vancouver Coastal Health, Vancouver, Canada b
A R T I C LE I N FO
A B S T R A C T
Keywords: Spinal cord injury Sympathetic preganglionic neurons Cardiovascular function Morphometry
Spinal cord injury (SCI) disrupts the supraspinal vasomotor pathways to sympathetic preganglionic neurons (SPNs) leading to impaired blood pressure (BP) control that often results in episodes of autonomic dysreflexia and orthostatic hypotension. The physiological cardiovascular consequences of SCI are largely attributed to the plastic changes in spinal SPNs induced by their partial deafferentation. While multiple studies have investigated the morphological changes in SPNs following SCI with contrasting reports. Here we investigated the morphological changes in SPNs rostral and caudal to a high thoracic (T3) SCI at 1-, 4- and 8-weeks post injury. SPNs were identified using Nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH- diaphorase) staining and were quantified for soma size and various dendritic measurements. We show that rostral to the lesion, soma size was increased at 1 week along with increased dendritic arbor. The total dendritic length was also increased at chronic stage (8 weeks post SCI). Caudal to the lesion, the soma size or dendritic lengths did not change with SCI. However, dendritic branching was enhanced within a week post SCI and remained elevated throughout the chronic stages. These findings demonstrate that SPNs undergo significant structural changes form sub-acute to chronic stages post-SCI that likely determines their functional consequences. These changes are discussed in context of physiological cardiovascular outcomes post-SCI.
1. Introduction In addition to sensory and motor impairments, SCI results in a myriad of debilitating autonomic dysfunctions that severely impact the survival and quality of life post injury (Hou and Rabchevsky, 2014; Krassioukov, 2009). With remarkable strides being made in acute care and management of other secondary conditions such as urinary tract infections, septicemia, renal dysfunction and pneumonia, cardiovascular dysfunction emerges as one of the leading causes of morbidity and mortality following SCI (Cragg et al., 2013; Garshick et al., 2005). Understandably, cardiovascular health is among the highest priorities for recovery in the individuals with SCI (Anderson, 2004; Hammell, 2010). In addition to having a consistently low resting blood pressure (BP), individuals with tetraplegia or high paraplegia (SCI above sixth thoracic segment; T6) frequently undergo dramatic BP fluctuations (Sachdeva et al., 2019). Within the same individual with SCI, while an orthostatic challenge (e.g., assuming an upright posture) can cause the systolic BP to drop down to 50 mmHg or lower (orthostatic
⁎
hypotension), a noxious or non-noxious stimulus originating below the spinal cord lesion (e.g., distended bladder) can trigger a sudden rise in BP reaching up to 300 mmHg (autonomic dysreflexia). These hypotensive and hypertensive crises are the result of either insufficient or excessive vasoconstriction respectively, primarily as a result of maladaptive changes in spinal SPNs below the lesion (Krassioukov, 2009). SPNs, located in four regions of spinal cord (the intermediolateral cell column, the intercalated nucleus, central autonomic nucleus and the lateral funiculus) between the T1 and L2 segments, determine the final sympathetic outflow from the central nervous system (CNS), regulating various autonomic functions including the BP (Llewellyn-Smith, 2009). Although cardiovascular dysfunction has been reported with SCI as low as T10 spinal segment (Gimovsky et al., 1985), it is most frequently observed in individuals with lesions above T6 segment, below which lie the SPNs responsible for modulating the highly compliant splanchnic and lower extremity blood vessels (i.e. the primary capacitance vascular bed). Despite complex pathophysiological alterations in central as well as peripheral components of the autonomic nervous system, cardiovascular dysfunction following SCI is largely attributed to the
Corresponding author at: ICORD-BSCC, 818 West 10th Avenue, Vancouver, BC V5Z 1M9, Canada. E-mail address: [email protected] (A.V. Krassioukov).
https://doi.org/10.1016/j.expneurol.2020.113235 Received 10 July 2019; Received in revised form 15 January 2020; Accepted 6 February 2020 Available online 07 February 2020 0014-4886/ © 2020 Elsevier Inc. All rights reserved.
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2.2. Tissue processing
changes resulting from decentralization of SPNs (Biering-Sorensen et al., 2017). Within the past two decades, a number of research groups, including ours have utilized human and rodent spinal cords to provide crucial insights into structural and functional maladaptations of SPNs, leading to abnormal sympathetic control of arterial pressure following SCI (Kalincik et al., 2010; Krassioukov et al., 1999; Krassioukov and Weaver, 1995, 1996; Lujan et al., 2010). However, a considerable lack of agreement exists among the specific findings reported in these studies e.g. temporal changes in soma size as well as dendritic arbor rostral and caudal to the injury site. A clear understanding of the time course of morphological changes in SPNs is important in unraveling of the mechanisms underlying autonomic cardiovascular dysfunction following SCI. In the present study we sought to address some of the discrepancies put forward across previous studies investigating the morphology of SPNs following SCI. Using a T3 complete transection model of SCI, we investigated the morphometric changes in a large population of SPNs in the spinal cord rostral and caudal to injury, by staining for NADPHdiaphorase. The results obtained in this study are discussed in context of previous literature.
At each experimental time point, the animals were euthanized with an overdose of chloral hydrate (Trichloroacetaldehyde monohydrate, 1 g/Kg, i.p.) and perfused transcardially with 150 ml of 0.1 M Phosphate buffer (pH 7.4), followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) (Krassioukov and Weaver, 1996). The spinal cord was removed and post-fixed in 4% paraformaldehyde for 12 h and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer for a minimum of 72 h (Sachdeva et al., 2016a). Thoracic spinal cord segments from C8 to T7 were embedded in Cryomatrix®, frozen in liquid nitrogen and stored at −80 °C prior to sectioning. The spinal cord segments were cut on a Cryostat® at 30 μm thick horizontal longitudinal sections, followed by mounting on Superfrost® Plus glass slides and stored at −20 °C. 2.3. Histochemical identification of SPNs using NADPH-diaphorase NADPH-diaphorase is a nitric oxide synthase enzyme constitutively expressed by spinal SPNs. This enzyme is capable of transferring hydrogen from NADPH to a soluble tetrazolium salt, converting it into insoluble, stable and distinctly visible formazan (Kluchova et al., 2001; Krassioukov and Weaver, 1996). Briefly, a hydrophobic barrier was made on the glass slide around the tissue using rubber cement (Elmer's®). Tissue sections were washed in 1 × phosphate buffer saline (PBS) solution twice for 10 min on a bench top orbital shaker. The tissue was incubated at 37 °C for 15 h in a solution of 5% nitro-blue tetrazolium chloride (NBT; ThermoFisher Scientific®), 2% NADPH (Sigma-Aldrich®) in 0.2% Triton-1 × PBS solution. Following incubation, rubber cement was removed and the slides were washed three times for 10 min in 1 × PBS solution (pH 7.4), and once in 100% alcohol for 15 min. The slides were air-dried for 2 h and cover-slipped using Cytoseal®. Sections were imaged under at 40 × magnification with an Aperio CS2 slide scanner.
2. Materials and methods Animal surgery and post-surgical care protocols were approved by the University of British Columbia Animal Care Committee and were performed in strict compliance with the policies established by the Canadian Council on Animal Care (institutional approval certificate number: A18–0183).
2.1. Spinal cord injury Twenty-four adult (~300 g), male Wistar rats (Harlan Laboratories, Indianapolis, IN) were divided across four groups (n = 6 each). Three groups received SCI and were euthanized at 1-, 4- and 8-weeks post injury. A fourth group received no SCI and served as an uninjured control. Surgical procedures and post-operative care were performed as published previously (Phillips et al., 2018; Ramsey et al., 2010). Starting three days prior to surgery, a subcutaneous injection of prophylactic enrofloxacin (Baytril; 10 mg/kg, Associated Veterinary Purchasing; AVP, Langley, Canada) was administered daily. On the day of the surgery, animals were anaesthetized with isoflurane using a 5% induction dose and maintained under surgical plane at 1.5–2%. Subcutaneous injections of enrofloxacin (10 mg/kg), buprenorphine (Temgesic®; 0.02 mg/kg, McGill University), and ketoprofen (Anafen®, 5 mg/kg, AVP) were administered pre-operatively and continued for 3 days post-surgery. Following a dorsal midline skin incision and blunt dissection of superficial muscles overlying C8 to T4, a laminectomy was performed at T2 vertebra to expose the T3 spinal cord segment. Dura mater was incised using a 30G needle and a complete transection was made using micro-scissors and gentle vacuum aspiration (Sachdeva et al., 2016b). Lesion completeness was confirmed using visual inspection and a pledget of Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) was placed between the stumps to achieve hemostasis. Dura was sutured close using 9–0 Prolene sutures (Sachdeva et al., 2016a). The muscle and skin were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively. Following surgery, animals received 5 ml subcutaneous injections of warm Lactated Ringer's solution (Baxter Corp., Canada) and allowed to recover in a temperature-controlled environment (Animal Intensive Care Unit, Los Angeles, CA, USA) before returning to home cages. Urinary bladders were manually expressed two to three times daily until spontaneous voiding returned (~ 10 days post injury).
2.4. Morphometric analysis and quantification Large scans of longitudinal spinal cord sections were visualized using the ImageScope software (Leica Biosystems). Individual neuron snapshots were randomly taken from two segments rostral and caudal to the lesion. Only neurons that met the criteria of having non-overlapping dendrites, distinguishable soma and uniform staining were included in quantification. A minimum of 20 neurons were analyzed per animal from at least 3 non-consecutive sections. Morphometric analysis was performed using the Fiji (Fiji Is Just ImageJ; https://fiji.sc/) application and was done blinded to the animal group and the neuron position in relation to the injury. All neuronal snapshots were blinded by a researcher unrelated to the study. Images were calibrated to scale and converted to 8-bit image type before analysis. The ImageJ freehand selection tool was used to trace the soma perimeter and data for soma area were calculated. Neurites were traced using the Simple Neurite Tracer Segmentation plugin (https://imagej. net/Simple_Neurite_Tracer) and analyzed using the Hessian-based analysis tools. The primary number of dendrites, total dendritic length summed from all dendrites and the maximum individual dendrite length per neuron were recorded. The Sholl analysis plugin (https:// imagej.net/Sholl_Analysis) was used to analyze all paths. Concentric rings with a radius step size of 10 μm were superimposed around the neuron soma center. Number of dendritic intersections at each concentric ring was summed and recorded as an indirect measure of dendritic arborisation (Lujan et al., 2010). 2.5. Statistical analyses GraphPad Prism software was used for statistical analyses. Two-way ANOVA (group vs. rostral/caudal location) was used to compare SPN 2
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Fig. 1. Identification of SPNs in spinal cord. (A) A longitudinal section of injured spinal cord stained for NADPH-diaphorase. Rectangular boxes indicate distinct regions containing SPNs magnified in (B) intermediolateral column, IML, (C) central autonomic area, CAA, and (D) intercalated nucleus, ICN. * indicates the lesion at T3.
Fig. 2. Quantification of soma size. (A) photomicrograph showing three NADPH-diaphorase-stained neurons showing cross sectional area of the soma highlighted using the freehand selection tool on ImageJ. (B) comparison of soma area in spinal SPNs rostral and caudal to injury showing a significant increase in soma size rostral to lesion at 1-week post-SCI. * indicates statistical significance, p < .05.
3.2. Soma size
soma size area, number of primary dendrites per neuron, maximum dendritic length per neuron and total dendritic length per neuron. For Sholl analysis results, two-way ANOVA (group vs. intersections) was performed to compare the number of dendritic intersections at consecutive 10 μm interval concentric rings. For all analyses, HolmŠídák's post hoc test was used for multiple comparisons. All data are reported as mean ± standard deviation (SD). In all analyses, data were considered statistically significance at p < .05.
Soma size was calculated as the 2-dimensional area by manually tracing the perimeter in ImageJ (Fig. 2A). Soma area was elevated at one-week post injury compared to uninjured controls in the neurons rostral to injury (Fig. 2B, Table 1). No differences were seen at other location or other timepoints. 3.3. Number of primary dendrites, maximum dendritic length, total dendritic length
3. Results
Following a semi-automated tracing of the dendrites, total dendritic length was summed from all dendrites and the maximum individual dendrite length per neuron were recorded (Fig. 3A). The number of primary dendrites were also counted manually for each neuron. Total dendritic length (Fig. 3B, Table 1) in the neurons rostral to the injury did not change at 1- and 4-weeks post SCI but was found to be significantly longer at 8-weeks post SCI. No effect of SCI was seen on the total dendritic length of the neurons below the lesion at any time points. Maximum individual dendritic length per neuron (Fig. 3C, Table 1) as well as the number of primary dendrites per neuron (Fig. 3D, Table 1) remained unchanged above and below the lesion following SCI.
3.1. Identification of SPNs Using the NADPH-diaphorase staining, spinal SPNs were identified in four distinct regions of a spinal cord section (see Fig. 1 for example). Majority of the SPNs were localized in close proximity to each other within the intermediolateral (IML) column at the junction of white and grey matter (Fig. 1B). Other spinal regions containing SPNs included the central autonomic area (CAA) near central canal (Fig. 1C), the intercalated nucleus (ICN; region between IML and CAA, Fig. 1D). A small proportion of neuronal cell bodies were also observed in the dorsolateral funiculus (not shown). No differences were observed between the right and left side of the spinal cord in terms of distribution or structure of neurons. Following the image acquisition, neurons within two segments rostral and caudal to injury (and corresponding segments from uninjured spinal cords) were randomly captured for analyses, while excluding the neurons with incomplete staining, truncated dendrites, excessive overlap with adjacent neurons. The neurons included in the quantification were free of bias towards the distinct regions i.e. IML, CAA and ICN.
3.4. Dendritic branching pattern: sholl analysis In order to assess the branching pattern of SPN dendrites, Sholl analysis (originally referred to as concentric shell method) was employed (Sholl, 1953). This quantitative analysis method allows the investigation of dendritic arborization using concentric circles as 3
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8 weeks
356.94 ± 97.00 420.13 ± 87.38 268.04 ± 62.71 2.89 ± 0.10
4 weeks
428.82 ± 119.88 323.27 ± 59.31 199.13 ± 22.58 2.53 ± 0.37
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coordinates of reference originating from the cell body. The extent of dendritic arbor indirectly represents the available postsynaptic space on the SPNs. For the present study, the concentric circles were calibrated at 10 μm step size and the number of dendritic intersections with circle at each step size were quantified (Fig. 4A). Rostral to the lesion, SCI led to an increase in the number of intersections (i.e. dendritic branching) at 1-week post injury and this effect was not significant at the later timepoints (Fig. 4B). More robust effect of SCI on dendritic branching was seen caudal to injury where SCI led to a significant increase in dendritic arbor at all three timepoints, suggesting a consistent increase in available postsynaptic space (Fig. 4C).
475.55 ± 70.33 358.82 ± 71.73 217.89 ± 47.91 2.58 ± 0.47 347.471 ± 127.5 446.19 ± 133.42 256.59 ± 36.85 2.52 ± 0.20 464.93 ± 65.33 640.52b ± 223.03 335.32 ± 167.92 3.11 ± 0.37
In order to understand the region-specific effect on SPN subpopulations, the morphometric characteristics of SPNs were further analyzed based on their localization in autonomic nuclei (Fig. 5). Within ICN, SPNs showed significant increase in soma size rostral to the lesion as early as 7 days post injury, which remained elevated for the remaining timepoints. Caudal to the lesion, ICN neurons showed an SCIdependent increase in soma size at 4 weeks post injury (Fig. 5A). A similar increase in soma size were also seen in SPNs within CAA caudal to the lesion at 7 days post injury (Fig. 5B). The maximum dendritic length of CAA neurons rostral to the injury was increased at 8 weeks post injury (Fig. 5C). No statistically significant differences were seen in the SPNs within IML (data not shown). 4. Discussion Using a high thoracic (T3) SCI, this study demonstrates the time course of morphological changes in SPNs rostral and caudal to injury. T3 complete SCI was utilized because this injury model decentralizes the majority of SPNs from the supraspinal cardiovascular control, while sparing two segments (T1 and T2) that provide sufficient SPNs to be studied as rostral, supraspinally connected controls. This injury model has been extensively characterized by our group and others in terms of cardiovascular dysfunction e.g. autonomic dysreflexia (Krassioukov et al., 2002; West et al., 2015b), cardiac structure and functional impairments (Poormasjedi-Meibod et al., 2019; Squair et al., 2018a), cerebrovascular (Phillips et al., 2016), gastrointestinal (Frias et al., 2018) as well as cardiometabolic dysfunctions (Inskip et al., 2010). Furthermore, using this model, we have also shown many promising treatment strategies resulting in significant cardiovascular recovery (Sachdeva, 2017; Squair et al., 2018b; West et al., 2015a). In this study, we observed that a high thoracic SCI leads to significant structural maladaptations in SPNs responsible for autonomic (e.g. cardiovascular) function. Rostral to the injury, SCI resulted in an early increase in soma size (at 1 week) that persisted for longer timepoints (i.e. 4 and 8 weeks timepoints were not different from 1 week). Dendrites on the other hand, did not change initially post injury but the total dendritic length was significantly greater at 8 weeks post SCI. Caudal to the injury, although no significant differences were seen in soma size or dendritic lengths, the dendrites showed significantly more branching compared to uninjured controls within a week post SCI and lasted for later time points. Interestingly, we also report region-specific responses to SCI by specific SPN subpopulations. Given the previously established location-specific neurochemical differences within SPNs (Hinrichs and Llewellyn-Smith, 2009), these results support the idea of structural and functional diversity observed within autonomic neurons (Llewellyn-Smith, 2009). This may have important implications in future research targeting the SPNs by therapeutic approaches for functional recovery (Kalincik et al., 2010). In context of previous pertinent literature, some similarities and differences exist. Our results are partially in agreement with those of Kalincik et al., which showed that both rostral and caudal to the SCI,
325.31 ± 130.07 364.21 ± 90.16 231.52 ± 41.45 2.69 ± 0.85 Soma size (μm ) Total dendritic length (μm) Maximum dendritic length (μm) Number of primary dendrites
Each cell represents mean ± SD for the group. a Indicates significant difference from the uninjured group. b Indicates significant difference from uninjured, 1 week and 4 weeks.
524.82 ± 121.42 379.90 ± 61.10 225.78 ± 34.06 2.85 ± 0.51
465.09 ± 116.57 330.37 ± 58.85 201.50 ± 50.35 2.69 ± 0.10
Uninjured 8 weeks 4 weeks 1 week Uninjured
2
Rostral to injury Analysis
Table 1 Morphometric measurements of SPNs following SCI.
a
Caudal to injury
1 week
3.5. Region-specific morphometric analyses
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Fig. 3. Quantification of number and length of dendrites. (A) photomicrograph showing dendrites traced using semi-automated Simple Neurite Tracer (ImageJ). (B) total dendritic length was significantly increased at 8-weeks post injury. No effect of SCI was seen on the maximum dendritic length (C) or the number of primary dendrites (D). * indicates statistical significance, p < .05.
Fig. 4. Quantification of dendritic branching. (A) An example image of a neuron analyzed via Sholl analysis, with 10 μm step size concentric rings superimposed around the soma (not shown to scale). Intersections within each concentric ring were summed as an indirect measurement of dendritic branching. (B) and (C) show significant increase in dendritic arborization of SPNs rostral and caudal to the lesion respectively. *, φ, and Ψ indicate statistical significance (p < .05) between uninjured control and 1-week, 4-weeks and 8-weeks respectively.
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Fig. 5. Region-specific morphometric analyses. (A) comparison of soma area within ICN SPNs rostral and caudal to injury showing a significant increase in soma size rostral to lesion at all time points post-SCI and at 4 weeks post SCI caudal to the lesion. (B) Within CAA, the soma area was increased caudal to the lesion at 7 days post injury. (C) Within CAA, the maximum dendritic length was increased rostral to the lesion at 8 weeks post injury. * indicates statistical significance, p < .05.
vasoconstriction in order to restore BP, despite a normal feedback via the arterial baroreflex system. Concomitantly on the other hand, the decentralized SPNs remain prone to be influenced by inputs arising from below the lesion. Consistent with previous studies, we show that this possibility may be further enhanced by increased dendritic branching of SPNs following SCI, putatively resulting in enhanced postsynaptic space. It is possible that increased dendritic arbor is accompanied by higher metabolic demand leading to increased soma size. Even though autonomic dysreflexia has also been reported in acute stages (Krassioukov et al., 2003), it is more commonly present in subacute and chronic stages- consistent with the aberrant sprouting of primary sensory afferents (Krenz and Weaver, 1998), suggesting that stimuli arising from primary sensory afferents below the lesion result in excitation of the SPNs leading to autonomic dysreflexia, most likely via multisynaptic long propriospinal pathways (Schramm, 2006).
soma size is increased, whereas dendritic length and number of primary dendrites are transiently increased (Kalincik et al., 2010). While we observed similar somal and dendritic effects rostral to injury, we did not see increased dendritic length caudal to injury. In our experiments, dendritic branching was significantly enhanced both rostral and caudal to injury but this parameter was unfortunately not investigated by Kalincik et al. A similar study by Lujan et al. also reported an SCI-dependent increase in soma size, dendritic length and branching rostral to injury in cardiac SPNs (Lujan et al., 2010). Our observations in SPNs rostral to SCI agree with this study despite the fact that this study analyzed a subpopulation of SPNs that project to stellate ganglia, in contrast to a more global population identified by NADPH-diaphorase staining. This study, however did not report the structural analyses in SPNs caudal to the injury site and hence that region cannot be compared. Finally, a discrepancy exists regarding whether there is an initial (1 week post-SCI) reduction in SPN soma size caudal to injury as reported previously (Krassioukov and Weaver, 1996). In contrast to this previous report, the present study agrees with the abovementioned studies by Kalincik et al. and Lujan et al. However, the initial discrepancy can be explained in at least two ways. Firstly, the previous study analyzed only a specific subpopulation of SPNs that project to adrenal medulla. It is possible that those SPNs behave differently and may not be an ideal representation of the total population of SPNs. This is partly supported by the observations that SPNs within different regions of spinal cord grey matter have specific structural and functional identities (Llewellyn-Smith, 2011; Wu et al., 2012). This has also been confirmed in the region-specific analyses in present study. Secondly, the reduction in soma size caudal to injury reported by Krassioukov and Weaver was shown in comparison to the SPNs rostral to injury, not to the SPNs in uninjured spinal cords (Krassioukov and Weaver, 1996). Therefore, this observed difference was likely due to the fact that the SPNs rostral to injury show a significant increase in soma size.
5. Summary The mechanisms underpinning cardiovascular consequences of SCI are enigmatic as the affected individuals can experience two opposite BP extremes within hours. Understanding the SCI-induced changes in key players involved in cardiovascular control is important in developing prevention/treatment strategies. We present and discuss temporal changes in SPNs following an experimental SCI that results in cardiovascular dysfunction and attempt to address some discrepancies across previous literature. A limitation of the present study is that although two-dimensional assessment of neuronal morphology is a standard procedure in the field, it does not account for dendritic arborization in z-dimension. Another inherent methodological limitation is that a large population of neurons, especially in the IML did not meet the inclusion criteria for analysis and were excluded. While orthostatic hypotension and autonomic dysreflexia are partly explained by SCIrelated changes in SPNs, it is important to mention that this discussion does not consider the numerous peripheral components contributing to cardiovascular dysfunction such as impaired renin-angiotensin system, cardiac deconditioning, reduction in plasma volume, fibrotic as well as hyper-responsiveness vasculature etc. A better understanding of these factors and their interplay is crucial towards a better understanding of cardiovascular impairments following SCI.
4.1. Implications for cardiovascular dysfunction Sympathetic activity and in turn, cardiovascular dysfunction following SCI is somewhat perplexing since it ranges from abnormally low to abnormally high BP within the same individual. SPNs being the final neurons determining the CNS sympathetic output are often regarded as key regulators of BP. It has been long known that SPNs do not exhibit intrinsic pacemaker potentials (Laskey and Polosa, 1988). Hence their activity in an intact nervous system is largely determined by spinal and supraspinal synaptic inputs (Guyenet, 2006). With the SCI-induced loss of supraspinal sympatho-modulatory input, the efferent sympathetic activity of SPNs is diminished. With significantly diminished tonic and reflex autonomic activity, individuals with a high-thoracic or cervical SCI experience a low resting BP as well as a substantial decline in BP while assuming an upright position (i.e. orthostatic hypotension). This is a consequence of the inability of supraspinal pathways to trigger
Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements The present study is supported by funds from Canadian Institutes of Health Research (CIHR) and Wings for Life Foundation. Dr. Krassioukov's laboratory is also supported by funds from the Heart and Stroke Foundation, Canadian Foundation for Innovation, BC Knowledge 6
Experimental Neurology 327 (2020) 113235
R. Sachdeva, et al.
Development Fund, Craig H. Neilsen Foundation and Seed grants from International Collaboration on Repair Discoveries (ICORD), supported by the Rick Hansen Foundation. Dr. Sachdeva is supported by Postdoctoral Fellowships from the Craig H. Neilsen Foundation, CIHR, Michael Smith Foundation for Health Research and University of British Columbia (Bluma Tischler Postdoctoral Fellowship). Mr. Marwaha's funding support is provided in part by the University of British Columbia Faculty of Medicine Summer Student Research Program. We would also like to thank Dr. David Granville and Mr. Yuan Jiang (ICORD) for their assistance with imaging experiments.
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