Rat motor neurons caudal to a rubrospinal tract (RST) transection remain viable

Accepted Manuscript Rat Motor Neurons Caudal to a Rubrospinal Tract (RST) Transection Remain Viable Brandon M. Wild, Rahul Mohan, Renée Morris PII: DOI: Reference:

S0306-4522(17)30648-6 http://dx.doi.org/10.1016/j.neuroscience.2017.09.013 NSC 18021

To appear in:

Neuroscience

Received Date: Accepted Date:

11 January 2017 6 September 2017

Please cite this article as: B.M. Wild, R. Mohan, R. Morris, Rat Motor Neurons Caudal to a Rubrospinal Tract (RST) Transection Remain Viable, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.09.013

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Rat Motor Neurons Caudal to a Rubrospinal Tract (RST) Transection Remain Viable Brandon M Wild, Rahul Mohan, Renée Morris Translational Neuroscience Facility School of Medical Science UNSW Medicine UNSW Australia Sydney, NSW 2052, Australia Correspondence: Renée Morris Translational Neuroscience Facility School of Medical Science UNSW Medicine UNSW Australia Sydney, NSW 2052, Australia [email protected] +61293858867

Keywords: motor neurons, rubrospinal tract, stereology, rat, cervical, lumbar

Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Abbreviations RST: Rubrospinal Tract DLF: Dorsolateral Funiculus RN: Red Nucleus SCI: Spinal Cord Injury

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Abstract In the rat, the rubrospinal tract (RST) is a descending motor pathway involved in the production of skilled reaching movement. The RST originates in the red nucleus in the midbrain and runs down the spinal cord in the lateral-most aspect of the dorsolateral funiculus (DLF). The RST makes monosynaptic contact with interneurons within the intermediate laminae of the cord, however a contingent of RST axons constitutes direct supraspinal input for spinal cord motor neurons. The current study investigated the effects of unilateral RST transection at cervical levels C3-4 on the population of motor neurons in both spinal segments C5-6 and L2-3. The total number of large, medium and small motor neurons in these segments was estimated with stereological techniques in both ventral horns at 1,3,7 and 14 days post-injury. In both spinal cord segments under investigation, no change was detected in mean number of motor neurons over time, in either ventral horns. That the loss of direct supraspinal input resulting from the RST transection does not affect the viability of motor neurons caudal to the injury indicates that these neurons have the potential to be re-innervated, should the RST injury be repaired.

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Introduction It has previously been established that damage to the spinal cord dorsolateral funiculus (DLF) impairs the rat’s ability to execute skilled reaching movement (Schrimsher and Reier, 1993; Muir et al., 2007; Stackhouse et al., 2008; Kanagal and Muir, 2009). Several ascending and descending pathways run within the DLF, including the Rubrospinal tract (RST) that occupies the lateral-most aspect of the funiculus. Injury restricted to the RST selectively abolishes the arpeggio movement, whereby the fingers pronate, in a way that resembles the arpeggio movement of a piano player (Morris et al., 2011; 2015). The RST’s well-established functional readout, together with its lateral position within the DLF, makes RST transection a distinctive rat model of spinal cord injury (SCI) involving only one single descending fibre tract (Morris and Whishaw, 2016). In the rat, the majority of RST axons terminate into the dorsal horn and the intermediate region of the ventral horn (Brown, 1974), however functional monosynaptic connections also exist between this descending fibre tract and spinal cord motor neurons. For instance, low threshold microsimulation of the Red Nucleus (RN), from which the RST originates yields short latency electromyographic responses in the distal forelimb, strongly suggesting that RST fibres do indeed make monosynaptic contact with spinal motor neurons (Küchler et al., 2002). Further, a tract tracing study involving injections of rabies virus in the rat forelimb have revealed intense trans-synaptically labelled neurons in the magnocellular subdivision of the RN, which gives rise to the RST (Ruigrok et al., 2008). It is important to note that there is no evidence of such monosynaptic connections between the corticospinal tract, i.e., the main input to spinal cord motor neurons in human- and nonhuman primates, and ventral horn motor neurons in the rat (Alstermark et al., 2004; Isa et al., 2007). Such direct connectivity between the RST and spinal cord motor neurons would allow the supraspinal motor commands to be forwarded, without modification, to these motor neurons. In this regard, the RST bears both anatomical and functional similarities to the main primate corticospinal motor pathway.

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RST transection creates neuronal atrophy rostrally in the midbrain RN, i.e., the origin of the tract (Barron et al., 1989; Ruitenberg et al., 2004). In looking at the effects of RST transection on the motor neurons below the site of injury, however, an exhaust of literature search yielded no results. It is likely that such surgical intervention would deprive the motor neurons caudal to the transection from an important source of supraspinal input. The aim of the present study was to determine the health of motor neurons below a RST transection, as knowledge regarding their fate would be imperative for the development of therapeutic strategies. In this study, we performed unilateral transection of the lateral-most component of the DLF, in which runs the RST, and assessed, by stereological quantification, its effect on the different motor neuron populations in the ventral horn of the spinal cord, immediately below the transection (at C5-6) as well as further caudally to the insult (at L2-3).

Methods Animals and Housing All experimental procedures complied with the Animal Care and Ethics Committee of the University of New South Wales (UNSW) and were performed in accordance with the National Health and Medical Research Council of Australia regulations for animal experimentation. A total of fifteen 8 week old adult female hooded rats (Rattus norvegicus, Long-Evans; Florey Institute, Monash University, Victoria, Australia), weighing approximately 230 grams at the time of surgery were used in this study. The rats were housed in groups of four in an animal holding room at the UNSW Biological Resource Centre (BRC) under a 12 hour light-dark cycle. Water and rat chow were provided ad libitum throughout the experiment. The rats were assigned into 5 groups: control (n=3) and animals that were subjected to RST transection and kept for: 1 day (n=3), 3 days (n=3), 7 days (n=3) and 14 days (n=3).

Surgical Procedure and Animal Care

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Surgeries were adapted from Morris et al. (2011). Briefly, transections were performed unilaterally on the right side of the spinal cord. The rats were injected intraperitoneally with a mixture of ketamine and xylazine (85mg/kg and 10mg/kg, respectively) to induce anaesthesia. The animals were fully monitored until they became ataxic and their corneal and pedal withdrawal reflexes were absent, at which point the hair overlying the cervical region of the spinal cord was shaved and the skin treated with 70% ethanol and Betadine, following which the animals were secured in a stereotaxic frame. A local analgesic (0.2mL Bupivacaine) was injected into the superficial dorsal muscles before an incision was made in the skin at the midline from the base of the skull to the level of the first thoracic vertebra. The paravertebral muscles were separated and retracted at the midline in order to expose the dorsal aspect of the vertebral column. A partial laminectomy was performed on the third and fourth cervical vertebra to expose the dorsolateral aspect of the spinal cord as well as the dorsal roots of C4 and C5. A small durotomy was then carried out and a transection of the lateral most part of the DLF was then performed between the two roots. In all occasions, care was taken in order to keep all the spinal roots intact as well as sparing the grey matter. Upon completion of the surgical procedure, the muscles were then sutured in layers with absorbent sutures and the skin incision was closed using surgical clips. A second dose of Bupivacaine was subsequently administered superficially on the skin at the site of the incision, and 5mL of 0.9% saline was delivered subcutaneously in order to prevent dehydration. The operated animals were closely monitored until they regained consciousness and were then moved to the holding area where weight and appearance were monitored daily to ensure proper recovery.

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Tissue Processing Perfusion Animals were sacrificed with a lethal dose of Lethabarb (300 mg/kg, Virbac, Sydney, New South Wales, Australia) and subjected to intracardial perfusion of 0.1M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) in 0.1M PB. Dissection Spinal segments from both the cervical and lumbar cord were dissected as per Tosolini and Morris, (2012) and Mohan et al., (2015). In order to dissect the cervical spinal cord, an incision was made in the skin over the superficial dorsal muscles and the paravertebral muscles overlaying the spinal cord were reflected to expose the vertebral column. The second cervical vertebra was first identified and then removed in order to expose the underlying spinal cord and dorsal roots, which were then marked with blue ink. This process was repeated with cervical vertebrae C3 to C6 in order to expose and mark the dorsal roots. The spinal cords were then cut transversely in situ into twosegment blocks (i.e. C3-4 and C5-6), and dissected out. In order to dissect out the lumbar spinal cord, an incision was made on the ventral aspect of the perfused rat, along the midline of the gut to remove the viscera and the posterior abdominal wall muscles. The caudal-most rib, T13, and its adjoining vertebra were identified and removed to expose the T13 ventral root. The vertebra rostral to T13 were then sequentially removed in order to follow the T13 ventral root to its entry point into the ventral horn of the spinal cord. The spinal root was then marked with blue ink at this junction. All the roots caudal to T13 were then individually identified and marked for easy identification. Spinal segment L2-3 was then cut transversely in situ as a two-segment block and dissected out. A superficial fiducial mark was made in the white matter midway between each two-segment block (i.e. C3-4, C5-6 and L2-3) to allow for easy orientation of the spinal cord segment. The blocks were then post-fixed overnight in a solution of 4% PFA in 0.1M PB, and cryoprotected in 30% sucrose solution in 0.1M PB for two days at 4°C. Histology 7

The spinal cord segments were frozen down and cut along the dorsoventral axis into 50µm sections using a cryostat. The sections from all segments were floated in microwell plates filled with 0.1M PB and subsequently mounted sequentially on microscope slides and allowed to dry overnight. All C5-6 and L2-3 tissue sections were then stained with Cresy-Violet. Sections from the C3-4, which contain the transections were stained with Luxol Fast Blue according to the standard Klüver-Barrera protocol (Klüver and Barrera, 1953) and counter-stained with Cresyl-Violet (Fig. 1).

Stereology Motor Neuron Counts All spinal segments were given random identities to ensure that the stereological aspect of the study was performed in a blinded fashion. Stereological quantification was performed with a Leica (Olympus BX51) microscope, using the automated Optical Fractionator Workflow probe of unbiased stereology (West, 1999) on the Stereoinvestigator software (MBF Biosciences). For large and medium-sized motor neurons, every fourth section was analysed. Every sixth section was analysed for small-sized motor neurons. In both instances, counting was commenced from a random starting section. An average of 18  2.11 and 16  2.87 (mean  SD) sections were analysed for spinal cord segment C5-6 and L2-3 respectively. For each selected section, the entire ipsilateral and contralateral ventral horns were contoured (see Fig. 2). The ventral horn was delineated from the level of the central canal to include the entirety of the ventral grey matter. Guard zones that extended to 10% of the thickness of each section were demarcated from the top and bottom of each section. For all the 3 motor neuron sub-types, a grid size of 100µm x 100µm was utilised. A counting frame of 60µm x 60µm was used to quantify large and medium-sized motor neurons whereas small motor neurons were counted with a 40µm x 40µm counting frame. The Stereoinvestigator software randomly places these specified counting frames throughout the contoured section of the ventral horn. An average of 1526  330 (mean  SD) counting frames were used to estimate the total number of large and medium motor neurons and 1291  384 for small motor neurons. 8

Counting Criteria All cells were counted using a x40 objective under bright field microscopy. The current study utilised an adaptation of the motor neuron classification system implemented in Bose et al.(2005) in order to decipher whether a cell can be identified as a motor neuron. Briefly, a cell was counted as a large motor neuron when the diameter of their cell bodies were greater than 34µm. Cells were classified as medium motor neurons when their cell body diameters ranged from 26-34µm. Cells were categorised as small motor neurons were identified as cells that possessed cell body diameters ranging from 11-25µm. Furthermore, only cells that possess a 1) well defined cell body, 2) a welldefined nucleus, and 3) a minimum of one clearly labelled Cresyl Violet-stained axon/dendrite. Further, all 3 features needed to be in focus within the dissector height for the cell to be included in the analysis. Care was taken to ensure that the cell body of each cell was required to lie either 1) within the counting frame, or 2) across the inclusion lines and not cross any of the exclusion lines. Within one Optical Fractionator Workflow, 80  7.21 (mean  SD) large and 120  9.57 medium motor neurons were counted in spinal cord segment C5-6. In spinal cord segment L2-3, 107  31.79 large motor neurons and 96  33.38 medium motor neurons were counted.

Estimation of total number of motor neurons The estimated total number of motor neurons (N) in the region of interest was determined by multiplying the sum of the number of motor neurons counted in the counting frame (Q-) by the thickness sampling fraction (tsf), area sampling fraction (asf) and the section sampling fraction (ssf).

Statistical Analysis

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The coefficient of error (CE) was utilised to assess the precision of the estimates derived from the sampling parameters. The CE was calculated using the formula described by Gundersen et al. (1999) and is itself a function of the total variance detected in the study (TotalVar) as well as the variance attributable to noise (s2). The mean CE of each studied group – large motor neurons, medium motor neurons and small motor neurons on both ipsilateral and contralateral side in both cervical and lumbar segments, were calculated and is represented in Table 1.

Motor neurons were counted for each rat and the mean number of motor neurons for each time point was analysed. Statistical comparisons between the mean counts of both ipsilateral and contralateral side were performed across all time points using two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-tests. Significance was set at p≤0.05. Effect size and power analysis were performed on both cervical and lumbar spinal cord segments.

Results Extent of injury Histological evaluation of the extent of the transections revealed that injuries were confined to the lateral aspect of the DLF. All transections were restricted to the white matter, displayed no impingements onto any aspect of the grey matter (see Fig. 1).

Difference in motor neuron population across all time points All parametric tests have satisfied all criteria for both equal variances and normal distribution. A two-way ANOVA demonstrated no significant statistical differences in the interaction between control and experimental animals with regards to the estimated population of large, medium and small motor neurons present in the spinal segments of interest across the different time points: 1

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day, 3 days, 7 days and 14 days post-surgery and between the ipsilateral and contralateral sides to the injury (see Table 1 and Fig. 3). Analysis of the mean number of large motor neurons within these segments revealed no statistical significant differences between the ipsilateral and contralateral side in both the cervical (p=0.993) and lumbar (p=0.981) segments over time. Analysis of the mean number of medium motor neurons in both cervical (p=0.954) and lumbar (p=0.875) spinal cord segments demonstrated no statistical significant differences between ipsilateral and contralateral sides to the injury over time. Bonferroni’s multiple comparison post-tests revealed no statistical significance for both ipsilateral and contralateral sides in both cervical and lumbar spinal cord segments over time. Effect size calculation revealed an average eta-squared (2) value of 0.32  .05 (mean  SEM) in the cervical spinal cord segment and 0.07  0.007 (mean  SEM) in the lumbar spinal cord segment. Power analysis of the mean estimated number of large and medium motor neurons revealed that a sample size of 51 for the cervical and 268 for lumbar spinal cord segments would be required to reject the null hypothesis.

Discussion In the intact central nervous system, inputs from higher brain centres devoted to the control of voluntary movement reach the spinal cord motor neurons that, upon excitation, trigger muscle contraction. After a spinal cord trauma, motor neurons below the injury are deprived of this supraspinal input, resulting in enduring paralysis. To date, however, little is known upon the way motor neurons respond to the removal of their supraspinal input from the brain. To our knowledge, this is the first study to assess the effects of the deprivation of direct supraspinal input, via the RST, on motor neurons both immediately caudal to the transection (at C5-6) as well as further downstream from the site of injury (L2-3). In this study, stereological analysis was carried out in order to decipher the effects of a RST transection on the population of large, medium and small motor neurons lying in the ventral horn at the above-mentioned levels across 1 to 14 days post11

injury. This analysis revealed no significant loss of neurons within the three different populations at both cervical and lumbar levels across all time points, on both the ipsilateral and contralateral sides to the injury. These results are exciting as they determine that these populations of motor neurons remain viable, despite the detection of a functional deficit, and therefore amenable to the therapeutic interventions that seek to re-establish the loss in functionality attributed by SCI.

Choice of spinal segments The integrity of the lateral aspect of the DLF, and more particularly of the RST, has been established to be essential to the execution of the arpeggio movement during skilled reaching (Whishaw et al., 1998; Morris et al., 2011). We have previously characterised the muscle-motor neuron topography in the rat forelimb (Tosolini and Morris, 2012). In this study, it was determined that Pronator Teres, the muscle involved in the execution of the pronation component of the arpeggio movement, is innervated by motor neurons spanning cervical segments C5 to C7 of the spinal cord. Therefore, motor neuron counts were performed on two-segment blocks that lie within this spinal cord region. Along with its role in skilled forelimb movement, the RST is also known to contribute to hindlimb locomotive activity. Locomotive deficits were observed for the hindlimb contralateral to a unilateral RN lesion (Muir and Whishaw, 2000) as well as ipsilateral to a unilateral (Webb and Muir, 2003) and bilateral (Muir et al., 2007) injury to the DLF where the RST is known to run. We therefore sought to determine the effects of a transection to the RST on the population of motor neurons that innervate Biceps Femoris and Vastus Medialis, which serve as extensors of the hip and knee respectively. These muscles comprise the two largest muscles of the lower limb and act on the proximal and lower leg respectively. We have previously established that Biceps Femoris and Vastus Medialis receive innervation from motor neuron columns spanning L2-3 of the rat spinal cord (Mohan et al., 2015) , and, as such, performed stereological analysis on these two segments of the lumbar spinal cord.

Comparison to Existing Literature 12

Several groups have studied the effects of SCI on motor neuron populations caudal to the injury site. After complete transections to the cord, Eidelberg et al., (1989) found significant decreases in the number of motor neurons caudal to the injury made at L4-5 from the first day following the trauma. Blunt contusion injury, on the other hand, results in a reduction in the number of motor neurons below the injury (Yong et al., 1998; Bose et al., 2005). Blunt contusion injuries are known to result primarily in the disruption of the grey matter, sparing a peripheral rim of white matter (Noble and Wrathall, 1985; Basso et al., 1996; Magnuson et al., 1999; Metz et al., 2000). The direct involvement of the grey matter in the above injury models could potentially be attributable to the changes observed in the motor neuron pools caudal to the injury. Contrastively, others have noted no significant loss of motor neurons distal to complete as well as partial transections (McBride and Feringa, 1992; Bjugn et al., 1997) in the rat. These findings corroborate findings in other species. In the cat for instance, Cook et al. (1951), found no reduction in motor neuron numbers following deprivation of supraspinal input. In human studies, Kaelan et al. (1988) found no loss in motor neuron populations distal to complete spinal cord injuries. These results taken together support the results obtained in the present study in that no differences in the population of motor neurons are observed following a transection to the RST.

Caveat The present study has investigated the effect of an RST transection on different motor neuron populations caudal to the injury from 1 day to 14 days post injury. Although no motor neuron loss was observed during this time frame, it is quite possible, that cell atrophy and/or death is induced could be induced at longer time points following injury. On a different note, the majority of RST axons have been shown to terminate onto interneurons (Brown, 1974; Antal et al., 1992). Interneurons are small, with cell body diameters that range from 12µm-20µm (Kubota and Kawaguchi, 2000). In the present study, a motor neuron classification system has been implemented in which small motor neurons are defined as having cell body diameters ranging from 11-25µm (Bose et al., 2005). As a result, the estimate of the population of small motor neurons reported here 13

could be confounded with the inclusion of interneurons found within the confines of the ventral horn. Consequently, the lack of a significant reduction in the number of motor neurons following the injury applies to interneurons as well. Immunoreactivity for parvalbumin or calbindin would be needed in order to differentiate between small motor neurons and interneurons.

Future Direction Large motor neurons can be further sub-classified into three types of motor neurons according to the contractile properties of the muscle fibres they innervate: slow-twitch fatigue-resistant (S) fibres, fast-twitch fatigable (FF) fibres and fast-twitch fatigue resistant (FR) fibres (Kanning et al., 2010). These large neurons vary in size depending on the type of muscle fibres they innervate: motor neurons that innervate slow-twitch fibres have smaller cell bodies and shorter axons, whilst motor neurons that innervate fast-twitch fibres have larger cell bodies and longer axons. After SCI, a shift in muscle fibre types from S fibres to FF fibres is observed in both humans and rats (Grimby et al., 1976; Lieber et al., 1986). Additional studies have also revealed electrophysiological changes within motor neurons following SCI (Cope et al., 1986; Hochman and McCrea, 1994). It would be worthwhile, therefore, to investigate changes in these parameters following injuries restricted to the DLF. RST fibres are known to make contact with motor neurons in the ventral horn directly as well as indirectly, by means of inter neurons. The current study examines the effect of a loss of supraspinal input onto motor neurons and interneurons present in the ventral horn only. It would be worthwhile examining the effects of the injury on interneurons between laminae V-VII (Antal et al., 1992), which are also known to receive input from the RST. The results from the present study would prove instrumental in filling the gap in knowledge regarding the fate of the motor neurons that have been deprived of their supraspinal input from the brain, as a result of a transection to the RST. It reveals that the these cells are not affected by atrophy and do not undergo cell death up to 14 days post-injury. Thus, if regeneration of the

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descending motor tracts could be achieved, these cells have the potential to be re-innervated and thus the functional deficit that is observed following injury could be potentially remediated.

Acknowledgements This work was funded by a National Health and Medical Research Centre (NHMRC) project grant and a Brain Foundation project grant to Renée Morris.

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Results Figure 1. Photomicrograph of a typical hemi-transverse section showing damage to the lateral aspect of the DLF. The arrows point to the limits of the transection. The transection includes the RST and is restricted to the white matter of the DLF, with no impingement onto any aspect of the grey matter. Figure 2 (A). Photomicrograph of Cresyl Violet stained hemi-transverse section through spinal cord segment C5-6. The red outline indicates the limits of the ventral horn that has been contoured using Stereoinvestigator software. All counting frames were constricted to this region. (B) Magnified view of the inset in (A) revealing Cresyl Violet stained motor neurons with a typical counting frame for reference. (O) shows a motor neuron that lies within the inclusion lines, depicted in green, and thus was included in the count. (X) shows a motor neuron lying across the exclusion lines, depicted in red, and thus was excluded from the count. Figure 3. The estimated mean number of (A) large and (B) medium motor neurons in C56 and (C) large and (D) medium motor neurons in L2-3, both ipsilateral and contralateral to the injury site for all time points. Error bars indicates the standard deviation of the mean. Table 1. Mean number of large, medium and small motor neurons for each time point on both ipsilateral and contralateral sides, as well as the associated mean coefficient of error (CE), based on the formula

and coefficient of variation (CV) for all studied

groups.

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Figure 1

Figure 2

Figure 3

Ipsilateral C5-6 Spinal Cord Segment Large Medium Small Control 1104 1794 13519 1 Day 1117 1672 13284 3 Days 1195 1669 12612 7 Days 1156 1793 14088 14 Days 1191 1652 13272 CE 0.11 0.09 0.06 CV 0.04 0.08 0.04 Contralateral C5-6 Spinal Cord Segment Control 1066 1789 13663 1 Day 1074 1590 13502 3 Days 1169 1603 12816 7 Days 1123 1817 14129 14 Days 1191 1581 13390 CE 0.11 0.09 0.06 CV 0.08 0.07 0.05 Ipsilateral L2-3 Spinal Cord Segment Control 1781 1610 10808 1 Day 1709 1589 10844 3 Days 1702 1542 10568 7 Days 1764 1582 10395 14 Days 1747 1529 10593 CE 0.10 0.11 0.07 CV 0.06 0.07 0.07 Contralateral L2-3 Spinal Cord Segment Control 1786 1609 9141 1 Day 1782 1666 9843 3 Days 1726 1653 10195 7 Days 1760 1598 10452 14 Days 1799 1695 10844 CE 0.10 0.10 0.07 CV 0.07 0.10 0.07 CE, Coefficient of Error (see text for calculation). CV, Coefficient of Variation

20

Highlights 

Spinal cord motor neurons do not undergo cell death up to 14 days following a loss in supraspinal input.



There are no differences in population in both ipsilateral and contralateral ventral horns after a unilateral transection.



Motor neuron populations in both spinal cord segments C5-6 and L2-3 showed no significant changes following injury.