A "Four Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases.

Background: We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods: XY rats were produced with an autosomal transgene of Sry , the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results: We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion: The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

EMG Testing throughout behavioral recovery after rat sciatic nerve crush injury results in exuberant motoneuron dendritic hypertrophy.

Background: Peripheral nerve injury (PNI) is the most common type of nerve trauma yet, while injured motoneurons exhibit a robust capacity for regeneration, behavioral recovery is protracted and typically poor. Neurotherapeutic approaches to PNI and repair have primarily focused on the enhancement of axonal regeneration, in terms of rate, axonal sprouting, and reconnection connectivity. Both electrical stimulation (ES) and treatment with androgens [e.g., testosterone propionate (TP)] have been demonstrated to enhance axonal sprouting, regeneration rate and functional recovery following PNI. To date, very little work has been done to examine the effects of ES and/or TP on dendritic morphology and organization within the spinal cord after PNI. Objective: The objective of the current study was to examine the impact of treatment with TP and ES, alone or in combination, on the dendritic arbor of spinal motoneurons after target disconnection via sciatic nerve crush injury in the rat. Methods: Rats received a crush injury to the sciatic nerve. Following injury, some animals received either (1) no further treatment beyond implantation with empty Silastic capsules, (2) electrical nerve stimulation immediately after injury, (3) implantation with Silastic capsules filled with TP, or (4) electrical nerve stimulation immediately after injury as well as implantation with TP. All of these groups of axotomized animals also received bi-weekly electromyography (EMG) testing. Additional groups of intact untreated animals as well as a group of injured animals who received no further treatment or EMG testing were also included. Eight weeks after injury, motoneurons innervating the anterior tibialis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Results: After nerve crush and ES and/or TP treatment, motoneurons innervating the anterior tibialis underwent marked dendritic hypertrophy. Surprisingly, this dendritic hypertrophy occurred in all animals receiving repeated bi-weekly EMG testing, regardless of treatment. When the EMG testing was eliminated, the dendritic arbor extent and distribution after nerve crush in the treated groups did not significantly differ from intact untreated animals. Conclusions: The ability of repeated EMG testing to so dramatically affect central plasticity following a peripheral nerve injury was unexpected. It was also unexpected that gonadal steroid hormones and/or ES, two neurotherapeutic approaches with demonstrated molecular/behavioral changes consistent with peripheral improvements in axonal repair and target reconnection, do not appear to impact central plasticity in a similar manner. The significance of peripheral EMG testing and resulting central plasticity reorganization remains to be determined.

Intraneural Topography of Rat Sciatic Axons: Implications for Polyethylene Glycol Fusion Peripheral Nerve Repair.

Peripheral nerve injuries are the most common type of nerve trauma. We have been working with a novel repair technique using a plasmalemmal fusogen, polyethylene glycol (PEG), to re-fuse the membranes of severed axons. PEG-fusion repair allows for immediate re-innervation of distal targets, prevents axonal degeneration, and improves behavioral recovery. PEG-fusion of severed axons is non-specific, and we have previously reported that following injury and PEG-fusion misconnections between spinal motoneurons and their distal targets were present. Surprisingly, appropriately paired proximal and distal motor axons were observed in all PEG-fused animals. We hypothesized that a topographic organization of axons contributing to the sciatic nerve could explain the incidence of appropriate connections. We traced the course of specific axon populations contributing to the sciatic nerve in young adult male and female rats. Following intraneural injection of Fast Blue into the tibial branch, labeled axons were confined to a discrete location throughout the course of the nerve. Following intramuscular injection of cholera toxin-conjugated horseradish peroxidase into the anterior tibialis, labeled axons were confined to a smaller but still discrete location throughout the nerve. In both cases, the relative locations of labeled axons were consistent bilaterally within animals, as well as across animals and sexes. Thus, the relatively consistent location of specific axon populations could allow for realignment of appropriate populations of axons, and enhanced behavioral recovery seen in PEG-fused animals. Knowing the organization of axons within the sciatic nerve promotes accurate territory realignment during repair, therefore aiding in recovery outcomes.

Inhibition of Cytosolic Phospholipase A2 Has Neuroprotective Effects on Motoneuron and Muscle Atrophy after Spinal Cord Injury.

Surviving motoneurons undergo dendritic atrophy after spinal cord injury (SCI), suggesting an important therapeutic target for neuroprotective strategies to improve recovery of function after SCI. Our previous studies showed that cytosolic phospholipase A2 (PLA2) may play an important role in the pathogenesis of SCI. In the present study, we investigated whether blocking cytosolic PLA2 (cPLA2) pharmacologically with arachidonyl trifluoromethyl ketone (ATK) or genetically using cPLA2 knockout (KO) mice attenuates motoneuron atrophy after SCI. C57BL/6 mice received either sham or contusive SCI at the T10 level. At 30 min after SCI, mice were treated with ATK or vehicle. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. ATK administration reduced percent lesion volume and increased percent volume of spared white matter, compared to the vehicle-treated control animals. SCI with or without ATK treatment had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with ATK. Similarly, vastus lateralis muscle weights of untreated SCI animals were smaller than those of sham surgery controls, and these reductions were prevented by ATK treatment. No effects on fiber cross-sectional areas, motor endplate area, or density were observed across treatment groups. Remarkably, genetically deleting cPLA2 in cPLA2 KO mice attenuated dendritic atrophy after SCI. These findings suggest that, after SCI, cord tissue damage and regressive changes in motoneuron and muscle morphology can be reduced by inhibition of cPLA2, further supporting a role for cPLA2 as a neurotherapeutic target for SCI treatment.

Exercise is neuroprotective on the morphology of somatic motoneurons following the death of neighboring motoneurons via androgen action at the target muscle.

Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that an androgen-dependent effect of exercise following injury can be neuroprotective against this dendritic atrophy. In this study, we explored where the necessary site of androgen action is for exercise-driven neuroprotective effects on induced dendritic atrophy. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected animals were given implants of the androgen receptor antagonist hydroxyflutamide, either directly at the adjacent vastus lateralis musculature ipsilateral to the saporin-injected vastus medialis or interscapularly as a systemic control. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the same vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Dendritic arbor lengths of saporin-injected animals allowed to exercise were significantly longer than those not allowed to exercise. Androgen receptor blockade locally at the vastus lateralis muscle prevented the protective effect of exercise. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites, which acts via androgen receptor action at the target muscle.

Exercise promotes recovery after motoneuron injury via hormonal mechanisms.

Injuries to spinal motoneurons manifest in a variety of forms, including damage to peripheral axons, neurodegenerative disease, or direct insult centrally. Such injuries produce a variety of negative structural and functional changes in both the directly affected and neighboring motoneurons. Exercise is a relatively simple behavioral intervention that has been demonstrated to protect against, and accelerate recovery from, these negative changes. In this article, we describe how exercise is neuroprotective for motoneurons, accelerating axon regeneration following axotomy and attenuating dendritic atrophy following the death of neighboring motoneurons. In both of these injury models, the positive effects of exercise have been found to be dependent on gonadal hormone action. Here we describe a model in which exercise, hormones, and brain-derived neurotrophic factor might all interact to produce neuroprotective effects on motoneuron structure following neural injury.

Behavioral recovery and spinal motoneuron remodeling after polyethylene glycol fusion repair of singly cut and ablated sciatic nerves.

Polyethylene glycol repair (PEG-fusion) of severed sciatic axons restores their axoplasmic and membrane continuity, prevents Wallerian degeneration, maintains muscle fiber innervation, and greatly improves recovery of voluntary behaviors. We examined alterations in spinal connectivity and motoneuron dendritic morphology as one potential mechanism for improved behavioral function after PEG-fusion. At 2-112 days after a single-cut or allograft PEG-fusion repair of transected or ablated sciatic nerves, the number, size, location, and morphology of motoneurons projecting to the tibialis anterior muscle were assessed by retrograde labeling. For both lesion types, labeled motoneurons were found in the appropriate original spinal segment, but also in inappropriate segments, indicating mis-pairings of proximal-distal segments of PEG-fused motor axons. Although the number and somal size of motoneurons was unaffected, dendritic distributions were altered, indicating that PEG-fusion preserves spinal motoneurons but reorganizes their connectivity. This spinal reorganization may contribute to the remarkable behavioral recovery seen after PEG-fusion repair.

Neuroprotective Effects of Exercise on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons.

Background. Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that treatment with androgens can be neuroprotective against this dendritic atrophy. Exercise has also been shown to be protective following a variety of neural injury models and, in some cases, is dependent on androgen action. Objective. In this study, we explored whether exercise shows the same neuroprotective effect on induced dendritic atrophy as that seen with androgen treatment. Methods. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in 3 dimensions. Results. Dendritic arbor lengths of animals allowed to exercise were significantly longer than those not allowed to exercise. Conclusions. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites comparable to that seen with exogenous androgen treatment.

Gonadal hormones differentially regulate sex-specific stress effects on glia in the medial prefrontal cortex.

Women are more susceptible to various stress-linked psychopathologies, including depression. Dysfunction of the medial prefrontal cortex (mPFC) has been implicated in depression, and studies indicate sex differences in stress effects on mPFC structure and function. For example, chronic stress induces dendritic atrophy in the mPFC in male rats, yet dendritic growth in females. Recent findings suggest glial pathways toward depression. Glia are highly responsive to neuronal activity and function as critical regulators of synaptic plasticity. Preclinical models demonstrate stress-induced microglial activation in mPFC in males, yet deactivation in females. By contrast, stress reduces astrocyte complexity in mPFC in male rats, whereas the effects in females are unknown. Glia possess receptors for most gonadal hormones and gonadal hormones are known to modulate neuronal activity. Thus, gonadal hormones represent a potential mechanism underlying sex differences in glia, as well as divergent stress effects. Therefore, we examined the role of gonadal hormones in sex-specific stress effects on neuronal activity (ie FosB/ ΔFosB induction) and glia in the mPFC. The findings obtained indicate greater microglial activation in mPFC in females and a greater astrocyte area in males. Basal astrocyte morphology is modulated by androgens, whereas androgens or oestrogens dampen the microglial state in males. Astrocyte morphology is associated with neuronal activity in both sexes, regardless of hormonal condition. Chronic stress induced astrocytic atrophy in males, yet hypertrophy in females, with gonadal hormones partly regulating this difference. Stress effects on microglia are oestradiol-dependent in females. Taken together, these data suggest sex-specific, gonadal hormone-dependent stress effects on astrocytes and microglia in the mPFC.

Neuroprotective Effects on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons: A Role for Microglia?

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone protects motoneurons from induced dendritic atrophy. We explored a potential mechanism for this induced atrophy and protection by testosterone, examining the microglial response to partial depletion of motoneurons. Motoneurons innervating the vastus medialis muscles of adult male rats were killed by intramuscular injection of cholera toxin-conjugated saporin; some saporin-injected rats were treated with testosterone. Microglia were later visualized via immunohistochemical staining, classified as monitoring or activated, and counted stereologically. Partial motoneuron depletion increased the number of activated microglia in the quadriceps motor pool, and this increase was attenuated with testosterone treatment. The attenuation in microglial response could reflect an effect of testosterone on suppressing microglia activation, potentially sparing motoneuron dendrites. Alternatively, testosterone could be neuroprotective, sparing motoneuron dendrites, secondarily resulting in reduced microglial activation. To discriminate between these hypotheses, following partial motoneuron depletion, rats were treated with minocycline to inhibit microglial activation. Motoneurons innervating the ipsilateral vastus lateralis muscle were later labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed. Reduction of microglial activation by minocycline did not prevent induced dendritic atrophy following partial motoneuron depletion. Further, reduction of microglial activation by minocycline treatment resulted in dendritic atrophy in intact animals. Together, these findings indicate that the neuroprotective effect of testosterone on dendrites following motoneuron death is not due to inhibiting microglial activation, and that microglial activity contributes to the normal maintenance of dendritic arbors.

Polyethylene glycol (PEG) and other bioactive solutions with neurorrhaphy for rapid and dramatic repair of peripheral nerve lesions by PEG-fusion.

BACKGROUND: Nervous system injuries in mammals often involve transection or segmental loss of peripheral nerves. Such injuries result in functional (behavioral) deficits poorly restored by naturally occurring 1-2 mm/d axonal outgrowths aided by primary repair or reconstruction. "Neurorrhaphy" or nerve repair joins severed connective tissues, but not severed cytoplasmic/plasmalemmal extensions (axons) within the tissue. NEW METHOD: PEG-fusion consists of neurorrhaphy combined with a well-defined sequence of four pharmaceutical agents in solution, one containing polyethylene glycol (PEG), applied directly to closely apposed viable ends of severed axons. RESULTS: PEG-fusion of rat sciatic nerves: (1) restores axonal continuity across coaptation site(s) within minutes, (2) prevents Wallerian degeneration of many distal severed axons, (3) preserves neuromuscular junctions, (4) prevents target muscle atrophy, (5) produces rapid and improved recovery of voluntary behaviors compared with neurorrhaphy alone, and (6) PEG-fused allografts are not rejected, despite no tissue-matching nor immunosuppression. COMPARISON WITH EXISTING METHODS: If PEG-fusion protocols are not correctly executed, the results are similar to that of neurorrhaphy alone: (1) axonal continuity across coaptation site(s) is not re-established, (2) Wallerian degeneration of all distal severed axons rapidly occurs, (3) neuromuscular junctions are non-functional, (4) target muscle atrophy begins within weeks, (5) recovery of voluntary behavior occurs, if ever, after months to levels well-below that observed in unoperated animals, and (6) allografts are either rejected or not well-accepted. CONCLUSION: PEG-fusion produces rapid and dramatic recovery of function following rat peripheral nerve injuries.

Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery.

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.

Protective effects of gonadal hormones on spinal motoneurons following spinal cord injury.

Spinal cord injury (SCI) results in lesions that destroy tissue and disrupt spinal tracts, producing deficits in locomotor and autonomic function. The majority of treatment strategies after SCI have concentrated on the damaged spinal cord, for example working to reduce lesion size or spread, or encouraging regrowth of severed descending axonal projections through the lesion, hoping to re-establish synaptic connectivity with caudal targets. In our work, we have focused on a novel target for treatment after SCI, surviving spinal motoneurons and their target musculature, with the hope of developing effective treatments to preserve or restore lost function following SCI. We previously demonstrated that motoneurons, and the muscles they innervate, show pronounced atrophy after SCI. Importantly, SCI-induced atrophy of motoneuron dendrites can be attenuated by treatment with gonadal hormones, testosterone and its active metabolites, estradiol and dihydrotestosterone. Similarly, SCI-induced reductions in muscle fiber cross-sectional areas can be prevented by treatment with androgens. Together, these findings suggest that regressive changes in motoneuron and muscle morphology seen after SCI can be ameliorated by treatment with gonadal hormones, further supporting a role for steroid hormones as neurotherapeutic agents in the injured nervous system.

Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats.

Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (∼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.

Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures, and behaviors lost after sciatic nerve transections in female rats.

Complete severance of major peripheral mixed sensory-motor nerve proximally in a mammalian limb produces immediate loss of action potential conduction and voluntary behaviors mediated by the severed distal axonal segments. These severed distal segments undergo Wallerian degeneration within days. Denervated muscles atrophy within weeks. Slowly regenerating (∼1 mm/day) outgrowths from surviving proximal stumps that often nonspecifically reinnervate denervated targets produce poor, if any, restoration of lost voluntary behaviors. In contrast, in this study using completely transected female rat sciatic axons as a model system, we provide extensive morphometric, immunohistochemical, electrophysiological, and behavioral data to show that these adverse outcomes are avoided by microsuturing closely apposed axonal cut ends (neurorrhaphy) and applying a sequence of well-specified solutions, one of which contains polyethylene glycol (PEG). This "PEG-fusion" procedure within minutes reestablishes axoplasmic and axolemmal continuity and signaling by nonspecifically fusing (connecting) closely apposed open ends of severed motor and/or sensory axons at the lesion site. These PEG-fused axons continue to conduct action potentials and generate muscle action potentials and muscle twitches for months and do not undergo Wallerian degeneration. Continuously innervated muscle fibers undergo much less atrophy compared with denervated muscle fibers. Dramatic behavioral recovery to near-unoperated levels occurs within days to weeks, almost certainly by activating many central nervous system and peripheral nervous system synaptic and other plasticities, some perhaps to a greater extent than most neuroscientists would expect. Negative control transections in which neurorrhaphy and all solutions except the PEG-containing solution are applied produce none of these remarkably fortuitous outcomes observed for PEG-fusion.

Conundrums and confusions regarding how polyethylene glycol-fusion produces excellent behavioral recovery after peripheral nerve injuries.

Current Neuroscience dogma holds that transections or ablations of a segment of peripheral nerves produce: (1) Immediate loss of axonal continuity, sensory signaling, and motor control; (2) Wallerian rapid (1-3 days) degeneration of severed distal axons, muscle atrophy, and poor behavioral recovery after many months (if ever, after ablations) by slowly-regenerating (1 mm/d), proximal-stump outgrowths that must specifically reinnervate denervated targets; (3) Poor acceptance of microsutured nerve allografts, even if tissue-matched and immune-suppressed. Repair of transections/ablations by neurorrhaphy and well-specified-sequences of PEG-fusion solutions (one containing polyethylene glycol, PEG) successfully address these problems. However, conundrums and confusions regarding unorthodox and dramatic results of PEG-fusion repair in animal model systems often lead to misunderstandings. For example, (1) Axonal continuity and signaling is re-established within minutes by non-specifically PEG-fusing (connecting) severed motor and sensory axons across each lesion site, but remarkable behavioral recovery to near-unoperated levels takes several weeks; (2) Many distal stumps of inappropriately-reconnected, PEG-fused axons do not ever (Wallerian) degenerate and continuously innervate muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (3) Host rats do not reject PEG-fused donor nerve allografts in a non-immuno-privileged environment with no tissue matching or immunosuppression; (4) PEG fuses apposed open axonal ends or seals each shut (thereby preventing PEG-fusion), depending on the experimental protocol; (5) PEG-fusion protocols produce similar results in animal model systems and early human case studies. Hence, iconoclastic PEG-fusion data appropriately understood might provoke a re-thinking of some Neuroscience dogma and a paradigm shift in clinical treatment of peripheral nerve injuries.

Protective Effects of Estradiol and Dihydrotestosterone following Spinal Cord Injury.

Spinal cord injury (SCI) results in lesions that destroy tissue and disrupt spinal tracts, producing deficits in locomotor and autonomic function. We previously demonstrated that motoneurons and the muscles they innervate show pronounced atrophy after SCI, and these changes are prevented by treatment with testosterone. Here, we assessed whether the testosterone active metabolites estradiol and dihydrotestosterone have similar protective effects after SCI. Young adult female rats received either sham or T9 spinal cord contusion injuries and were treated with estradiol, dihydrotestosterone, both, or nothing via Silastic capsules. Basso-Beattie-Bresnahan locomotor testing was performed weekly and voiding behavior was assessed at 3 weeks post-injury. Four weeks after SCI, lesion volume and tissue sparing, quadriceps muscle fiber cross-sectional area, and motoneuron dendritic morphology were assessed. Spontaneous locomotor behavior improved after SCI, but hormone treatments had no effect. Voiding behavior was disrupted after SCI, but was significantly improved by treatment with either estradiol or dihydrotestosterone; combined treatment was maximally effective. Treatment with estradiol reduced lesion volume, but dihydrotestosterone alone and estradiol combined with dihydrotestosterone were ineffective. SCI-induced decreases in motoneuron dendritic length were attenuated by all hormone treatments. SCI-induced reductions in muscle fiber cross-sectional areas were prevented by treatment with either dihydrotestosterone or estradiol combined with dihydrotestosterone, but estradiol treatment was ineffective. These findings suggest that deficits in micturition and regressive changes in motoneuron and muscle morphology seen after SCI are ameliorated by treatment with estradiol or dihydrotestosterone, further supporting a role for steroid hormones as neurotherapeutic agents in the injured nervous system.

Hormonal Treatment Effects on the Cross-sectional Area of Pubococcygeus Muscle Fibers After Denervation and Castration in Male Rats.

We explore the interaction of muscle innervation and gonadal hormone action in the pubococcygeus muscle (Pcm) after castration and hormone replacement. Male Wistar rats were castrated and the Pcm was unilaterally denervated; after 2 or 6 weeks, the cross-sectional area (CSA) of Pcm fibers was assessed. Additional groups of castrated rats were used to examine the effects of hormone replacement. At 2 weeks post surgeries, rats were implanted with Silastic capsules containing either dihydrotestosterone (DHT), estradiol benzoate (EB) or both hormones, and the CSA of Pcm fibers was assessed after 4 weeks of hormone treatment. At 2 weeks post surgeries, gonadectomy without hormone replacement resulted in reductions in the CSA of Pcm fibers, and denervation combined with castration increased the magnitude of this effect; further reductions in CSA were present at 6 weeks post surgeries, but again denervation combined with castration increased the magnitude of this effect. Hormone replacement with DHT resulted in hypertrophy in the CSA of nondenervated muscles compared to those of intact normal males, but this effect was attenuated in denervated muscles. Hormone replacement with EB treatment prevented further castration-induced reductions in CSA of nondenervated muscles, but denervation prevented this effect. Similar to that seen with treatment with EB alone, combined treatment with both DHT and EB prevented further reductions in CSA of Pcm fibers in nondenervated muscles, but again denervation attenuated this effect. Thus, while hormone replacement can reverse or prevent further castration-induced atrophy of Pcm fibers, these effects are dependent on muscle innervation. Anat Rec, 300:1327-1335, 2017. © 2017 Wiley Periodicals, Inc.