Delayed Systemic Treatment with Cannabinoid Receptor 2 Agonist Mitigates Spinal Cord Injury-Induced Osteoporosis More Than Acute Treatment Directly after Injury.

Nearly all persons with spinal cord injury (SCI) will develop osteoporosis following injury, and further, up to 50% of all persons with SCI will sustain a fracture during their lives. The unique mechanisms driving osteoporosis following SCI remain unknown. The cannabinoid system modulation of bone metabolism through cannabinoid 1/2 (CB1/2) has been of increasing interest for the preservation of bone mass and density in models of osteoporosis. Using a thoracic vertebral level 8 (T8) complete transection in a mouse model, we performed daily treatment with a selective CB2 receptor agonist, HU308, compared with SCI-vehicle-treated and naïve control animals either immediately after injury for 40 days, or in a delayed paradigm, following 3 months after injury. The goal was to prevent or potentially reverse SCI-induced osteoporosis. In the acute phase, administration of the CB2 agonist was not able to preserve the rapid loss of cancellous bone. In the delayed-treatment paradigm, in cortical bone, HU308 increased cortical-area to total-area ratio and periosteal perimeter in the femur, and improved bone density in the distal femur and proximal tibia. Further, we report changes to the metaphyseal periosteum with increased presence of adipocyte and fat mass in the periosteum of SCI animals, which was not present in naïve animals. The layer of fat increased markedly in HU308-treated animals compared with SCI-vehicle-treated animals. Overall, these data show that CB2 agonism targets a number of cell types that can influence overall bone quality.

P-glycoprotein Expression Is Upregulated in a Pre-Clinical Model of Traumatic Brain Injury.

Athletes participating in contact sports are at risk for sustaining repeat mild traumatic brain injury (rmTBI). Unfortunately, no pharmacological treatment to lessen the pathophysiology of brain injury has received U.S. Food and Drug Administration (FDA) approval. One hurdle to overcome for potential candidate agents to reach effective therapeutic concentrations in the brain is the blood-brain barrier (BBB). Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), line the luminal membrane of the brain capillary endothelium facing the vascular space. Although these transporters serve to protect the central nervous system (CNS) from damage by effluxing neurotoxicants before they can reach the brain, they may also limit the accumulation of therapeutic drugs in the brain parenchyma. Thus, increased Pgp expression following brain injury may result in reduced brain availability of therapeutic agents. We therefore questioned if repeat concussive injury increases Pgp expression in the brain. To answer this question, we used a rodent model of repeat mild closed head injury (rmCHI) and examined the messenger RNA (mRN) and protein expression of both isoforms of rodent Pgp (Abcb1a and Abcb1b). Compared with sham-operated controls (n = 5), the mRNA levels of both Abcb1a and Abcb1b were found to be increased in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury. Using a validated antibody, we show increased immunolabeling for Pgp in the dorsal cortex at day 5 and in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury compared with sham controls (n = 6). Taken together, these results suggest that increased expression of Pgp after rmCHI may reduce the brain accumulation of therapeutic drugs that are Pgp substrates. It is plausible that including a Pgp inhibitor with a candidate therapeutic agent may be an effective approach to treat the pathophysiology of rmCHI.

Acute damage to the blood­brain barrier and perineuronal net integrity in a clinically-relevant rat model of traumatic brain injury.

Closed-head, frontal impacts in which the brain undergoes both lateral and rotational acceleration comprise the majority of human traumatic brain injury (TBI). Here, we utilize a clinically relevant model to examine the effects of a single concussion on aspects of brain integrity: the blood-brain barrier, the perineuronal nets (PNNs), and diffuse axonal injury. Adult, male Sprague-Dawley rats received either a frontal, closed-head concussive TBI, or no injury and were evaluated at 1- or 7-day post-injury. Using immunolabeling for albumin, we observed a significant increase in the permeability of the blood-brain barrier at 1-, but not 7-day post-injury. Breakdown of the PNN, as measured by the binding of wisteria floribunda, was observed at 1-day post-injury in the dorsal, lateral, and ventral cortices. This difference was resolved at 7-day. Finally, axonal injury was identified at both 1- and 7-day post-injury. This preclinical model of closed-head, frontal TBI presents a useful tool with which to understand better the acute pathophysiology of a single, frontal TBI.

Energy balance following diets of varying fat content: metabolic dysregulation in a rodent model of spinal cord contusion.

Within the spinal cord injured (SCI) population, metabolic dysfunction may be exacerbated. Models of cord injury coupled with metabolic stressors have translational relevance to understand disease progression in this population. In the present study, we used a rat model of thoracic SCI at level T10 (tSCI) and administered diets comprised of either 9% or 40% butterfat to create a unique model system to understand the physiology of weight regulation following cord injury. SCI rats that recovered on chow for 28 days had reduced body mass, lean mass, and reduced fat mass but no differences in percentage of lean or fat mass composition. Following 12 weeks on either low-fat diet (LFD) or high-fat diet (HFD), SCI rats maintained on LFD did not gain weight at the same rate as SCI animals maintained on HFD. LFD-SCI had reduced feed conversion efficiency in comparison to Sham-LFD whereas tSCI-HFD were equivalent to Sham-HFD rats. Although SCI rats still maintained lower lean body mass, by the end of the study HFD-fed rats had higher body fat percentage than LFD-fed rats. Macronutrient selection testing demonstrated SCI rats had a significant preference for protein over Sham rats. Analysis of metabolic cage activity showed tSCI rats had elevated energy expenditure, despite reduced locomotor activity. Muscle triglycerides and cholesterol were reduced only in LFD-tSCI rats. These data suggest that consumption of HFD by tSCI rats alters the trajectory of metabolic dysfunction in the context of spinal cord disease progression.

RhoA-ROCK Inhibition Reverses Synaptic Remodeling and Motor and Cognitive Deficits Caused by Traumatic Brain Injury.

Traumatic brain injury (TBI) causes extensive neural damage, often resulting in long-term cognitive impairments. Unfortunately, effective treatments for TBI remain elusive. The RhoA-ROCK signaling pathway is a potential therapeutic target since it is activated by TBI and can promote the retraction of dendritic spines/synapses, which are critical for information processing and memory storage. To test this hypothesis, RhoA-ROCK signaling was blocked by RhoA deletion from postnatal neurons or treatment with the ROCK inhibitor fasudil. We found that TBI impairs both motor and cognitive performance and inhibiting RhoA-ROCK signaling alleviates these deficits. Moreover, RhoA-ROCK inhibition prevents TBI-induced spine remodeling and mature spine loss. These data argue that TBI elicits pathological spine remodeling that contributes to behavioral deficits by altering synaptic connections, and RhoA-ROCK inhibition enhances functional recovery by blocking this detrimental effect. As fasudil has been safely used in humans, our results suggest that it could be repurposed to treat TBI.

Chronic spinal cord changes in a high-fat diet-fed male rat model of thoracic spinal contusion.

Individuals that suffer injury to the spinal cord can result in long-term, debilitating sequelae. Spinal cord-injured patients have increased risk for the development of metabolic disease, which can further hinder the effectiveness of treatments to rehabilitate the cord and improve quality of life. In the present study, we sought to understand the impact of high-fat diet (HFD)-induced obesity on spinal cord injury (SCI) by examining transcriptome changes in the area of the injury and rostral and caudal to site of damage 12 wk after injury. Adult, male Long-Evans rats received either thoracic level contusion of the spinal cord or sham laminectomy and then were allowed to recover on normal rat chow for 4 wk and further on HFD for an additional 8 wk. Spinal cord tissues harvested from the rats were processed for Affymetrix microarray and further transcriptomic analysis. Diverse changes in gene expression were identified in the injured cord in genes such as MMP12, APOC4, GPNMB, and IGF1 and 2. The greatest signaling changes occurred in pathways involved in cholesterol biosynthesis and immune cell trafficking. Together, the cord changes in the chronically obese rat following thoracic SCI reveal further potential targets for therapy. These could be further explored as they overlap with genes involved in metabolic disease.

Cryopreserved human umbilical cord versus biocellulose film for prenatal spina bifida repair in a physiologic rat model.

BACKGROUND: Prenatal spina bifida (SB) repair with a regenerative patch may improve neurological outcomes by decreasing inflammatory scarring. OBJECTIVE: This study aims to compare cryopreserved human umbilical cord (HUC) and biocellulose film (BCF) patches sutured over SB lesions for regeneration of native cells and inflammatory response. STUDY DESIGN: Sprague-Dawley rats were gavaged with retinoic acid (RA) on embryonic day 10 to induce SB. Hysterotomy was performed on embryonic day 20 and on HUC or BCF patches sutured over the defect. Pups were harvested 30 to 34 h later, and hematoxylin and eosin staining and Trichrome staining assessed basic cellular migration. Immunohistochemistry demonstrated the exact nature of the cellular migration. Patches and surrounding exudates were evaluated with microscopy and cells quantified. RESULTS: Histology showed cellular migration with all HUC patches compared with none with BCF patches. Epithelial cells were noted migrating over the dorsal HUC surface, astrocytes were noted along the HUC surface adjacent to the lesion, and endothelial cells were noted within the HUC. HUC patches showed minimal inflammatory cells. Exudates surrounding the HUC patches had fewer inflammatory cells than exudates around BCF patches. CONCLUSION: HUC promotes cellular migration of native cells with minimal inflammatory response compared with BCF. HUC may be the superior patch material for prenatal SB repair. © 2017 John Wiley & Sons, Ltd.

Changes in Gene Expression and Metabolism in the Testes of the Rat following Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating changes to almost all aspects of a patient's life. In addition to a permanent loss of sensory and motor function, males also will frequently exhibit a profound loss of fertility through poorly understood mechanisms. We demonstrate that SCI causes measureable pathology in the testis both acutely (24 h) and chronically up to 1.5 years post-injury, leading to loss in sperm motility and viability. SCI has been shown in humans and rats to induce leukocytospermia, with the presence of inflammatory cytokines, anti-sperm antibodies, and reactive oxygen species found within the ejaculate. Using messenger RNA and metabolomic assessments, we describe molecular and cellular changes that occur within the testis of adult rats over an acute to chronic time period. From 24 h, 72 h, 28 days, and 90 days post-SCI, the testis reveal a distinct time course of pathological events. The testis show an acute drop in normal sexual organ processes, including testosterone production, and establishment of a pro-inflammatory environment. This is followed by a subacute initiation of an innate immune response and loss of cell cycle regulation, possibly due to apoptosis within the seminiferous tubules. At 1.5 years post-SCI, there is a chronic low level immune response as evidenced by an elevation in T cells. These data suggest that SCI elicits a wide range of pathological processes within the testes, the actions of which are not restricted to the acute phase of injury but rather extend chronically, potentially through the lifetime of the subject. The multiplicity of these pathological events suggest a single therapeutic intervention is unlikely to be successful.

Human Neural Stem Cell Transplantation-Mediated Alteration of Microglial/Macrophage Phenotypes after Traumatic Brain Injury.

Neural stem cells (NSCs) promote recovery from brain trauma, but neuronal replacement is unlikely the sole underlying mechanism. We hypothesize that grafted NSCs enhance neural repair at least partially through modulating the host immune response after traumatic brain injury (TBI). C57BL/6 mice were intracerebrally injected with primed human NSCs (hNSCs) or vehicle 24 h after a severe controlled cortical impact injury. Six days after transplantation, brain tissues were collected for Western blot and immunohistochemical analyses. Observations included indicators of microglia/macrophage activation, M1 and M2 phenotypes, axonal injury detected by amyloid precursor protein (APP), lesion size, and the fate of grafted hNSCs. Animals receiving hNSC transplantation did not show significant decreases of brain lesion volumes compared to transplantation procedures with vehicle alone, but did show significantly reduced injury-dependent accumulation of APP. Furthermore, intracerebral transplantation of hNSCs reduced microglial activation as shown by a diminished intensity of Iba1 immunostaining and a transition of microglia/macrophages toward the M2 anti-inflammatory phenotype. The latter was represented by an increase in the brain M2/M1 ratio and increases of M2 microglial proteins. These phenotypic switches were accompanied by the increased expression of anti-inflammatory interleukin-4 receptor α and decreased proinflammatory interferon-γ receptor ß. Finally, grafted hNSCs mainly differentiated into neurons and were phagocytized by either M1 or M2 microglia/macrophages. Thus, intracerebral transplantation of primed hNSCs efficiently leads host microglia/macrophages toward an anti-inflammatory phenotype that presumably contributes to stem cell-mediated neuroprotective effects after severe TBI in mice.

Minocycline plus N-acetylcysteine synergize to modulate inflammation and prevent cognitive and memory deficits in a rat model of mild traumatic brain injury.

Traumatic brain injury (TBI) differs in severity from severe to mild. This study examined whether a combination of the drugs minocycline (MINO) plus N-acetylcysteine (NAC) produces behavioral and histological improvements in a mild version of the controlled cortical impact model of TBI (mCCI). Following mCCI, rats acquired an active place avoidance task by learning the location of a stationary shock zone on a rotating arena. Rats acquired this task with a training protocol using a 10-minute intertrial interval. Mildly injured rats had an apparent deficit in long-term memory since they did not acquire the task when the intertrial interval was increased to 24 h. Mildly injured rats also had an apparent deficit in set shifting since, after successfully learning one shock zone location they did not learn the location of a second shock zone. MINO plus NAC synergistically limited these behavioral deficits in long-term memory and set shifting. mCCI also produced neuroinflammation at the impact site and at distal white matter tracts including the corpus callosum. At the impact site, MINO plus NAC attenuated CD68-expressing phagocytic microglia without altering neutrophil infiltration or astrocyte activation. The drugs had no effect on astrocyte activation in the corpus callosum or hippocampus. In the corpus callosum, MINO plus NAC decreased CD68 expression yet increased overall microglial activation as measured by Iba-1. MINO plus NAC acted synergistically to increase Iba-1 expression since MINO alone suppressed expression and NAC alone had no effect. Despite the known anti-inflammatory actions of the individual drugs, MINO plus NAC appeared to modulate, rather than suppress neuroinflammation. This modulation of neuroinflammation may underlie the synergistic improvement in memory and set-shifting by the drug combination after mCCI.

Licofelone modulates neuroinflammation and attenuates mechanical hypersensitivity in the chronic phase of spinal cord injury.

Inflammation is a major factor shaping outcome during the early, acute phase of traumatic spinal cord injury (SCI). It is known that pro-inflammatory signaling within the injured spinal cord drives pathological alterations in neurosensory processing and shapes functional outcome early after injury. However, it is unclear whether inflammation persists into the chronic phase of injury or shapes sensory processing long after injury. To investigate these possibilities, we have performed biochemical and behavioral assessments 9 months after moderate thoracic spinal contusion injury in the rat. We have found that levels of the pro-inflammatory lipid mediators leukotriene B4 and prostaglandin E2 are elevated in the chronic spinal cord lesion site. Additionally, using metabolomic profiling, we have detected elevated levels of pro-oxidative and inflammatory metabolites, along with alterations in multiple biological pathways within the chronic lesion site. We found that 28 d treatment of chronically injured rats with the dual COX/5-LOX inhibitor licofelone elevated levels of endogenous anti-oxidant and anti-inflammatory metabolites within the lesion site. Furthermore, licofelone treatment reduced hypersensitivity of hindpaws to mechanical, but not thermal, stimulation, indicating that mechanical sensitivity is modulated by pro-inflammatory signaling in the chronic phase of injury. Together, these findings provide novel evidence of inflammation and oxidative stress within spinal cord tissue far into the chronic phase of SCI, and demonstrate a role for inflammatory modulation of mechanical sensitivity in the chronic phase of injury.

Treatment of mild traumatic brain injury with an erythropoietin-mimetic peptide.

Mild traumatic brain injury (mTBI) results in an estimated 75-90% of the 1.7 million TBI-related emergency room visits each year. Post-concussion symptoms, which can include impaired memory problems, may persist for prolonged periods of time in a fraction of these cases. The purpose of this study was to determine if an erythropoietin-mimetic peptide, pyroglutamate helix B surface peptide (pHBSP), would improve neurological outcomes following mTBI. Sixty-four rats were randomly assigned to pHBSP or control (inactive peptide) 30 µg/kg IP every 12 h for 3 days, starting at either 1 hour (early treatment) or 24 h (delayed treatment), after mTBI (cortical impact injury 3 m/sec, 2.5 mm deformation). Treatment with pHBSP resulted in significantly improved performance on the Morris water maze task. Rats that received pHBSP required 22.3±1.3 sec to find the platform, compared to 26.3±1.3 sec in control rats (p=0.022). The rats that received pHBSP also traveled a significantly shorter distance to get to the platform, 5.0±0.3 meters, compared to 6.1±0.3 meters in control rats (p=0.019). Motor tasks were only transiently impaired in this mTBI model, and no treatment effect on motor performance was observed with pHBSP. Despite the minimal tissue injury with this mTBI model, there was significant activation of inflammatory cells identified by labeling with CD68, which was reduced in the pHBSP-treated animals. The results suggest that pHBSP may improve cognitive function following mTBI.

Analysis of functional pathways altered after mild traumatic brain injury.

Concussive injury (or mild traumatic brain injury; mTBI) can exhibit features of focal or diffuse injury patterns. We compared and contrasted the cellular and molecular responses after mild controlled cortical impact (mCCI; a focal injury) or fluid percussion injury (FPI; a diffuse injury) in rats. The rationale for this comparative analysis was to investigate the brain's response to mild diffuse versus mild focal injury to identify common molecular changes triggered by these injury modalities and to determine the functional pathways altered after injury that may provide novel targets for therapeutic intervention. Microarrays containing probes against 21,792 unique messenger RNAs (mRNAs) were used to investigate the changes in cortical mRNA expression levels at 3 and 24 h postinjury. Of the 354 mRNAs with significantly altered expression levels after mCCI, over 89% (316 mRNAs) were also contained within the mild FPI (mFPI) data set. However, mFPI initiated a more widespread molecular response, with over 2300 mRNAs differentially expressed. Bioinformatic analysis of annotated gene ontology molecular function and biological pathway terms showed a significant overrepresentation of genes belonging to inflammation, stress, and signaling categories in both data sets. We therefore examined changes in the protein levels of a panel of 23 cytokines and chemokines in cortical extracts using a Luminex-based bead immunoassay and detected significant increases in macrophage inflammatory protein (MIP)-1α (CCL3), GRO-KC (CXCL1), interleukin (IL)-1α, IL-1ß, and IL-6. Immunohistochemical localization of MIP-1α and IL-1ß showed marked increases at 3 h postinjury in the cortical vasculature and microglia, respectively, that were largely resolved by 24 h postinjury. Our findings demonstrate that both focal and diffuse mTBI trigger many shared pathobiological processes (e.g., inflammatory responses) that could be targeted for mechanism-based therapeutic interventions.

The dual cyclooxygenase/5-lipoxygenase inhibitor licofelone attenuates p-glycoprotein-mediated drug resistance in the injured spinal cord.

There are currently no proven effective treatments that can improve recovery of function in spinal cord injury (SCI) patients. Many therapeutic compounds have shown promise in pre-clinical studies, but clinical trials have been largely unsuccessful. P-glycoprotein (Pgp, Abcb1b) is a drug efflux transporter of the blood-spinal cord barrier that limits spinal cord penetration of blood-borne xenobiotics. Pathological Pgp upregulation in diseases such as cancer causes heightened resistance to a broad variety of therapeutic drugs. Importantly, several drugs that have been evaluated for the treatment of SCI, such as riluzole, are known substrates of Pgp. We therefore examined whether Pgp-mediated pharmacoresistance diminishes delivery of riluzole to the injured spinal cord. Following moderate contusion injury at T10 in male Sprague-Dawley rats, we observed a progressive, spatial spread of increased Pgp expression from 3 days to 10 months post-SCI. Spinal cord uptake of i.p.-delivered riluzole was significantly reduced following SCI in wild type but not Abcb1a-knockout rats, highlighting a critical role for Pgp in mediating drug resistance following SCI. Because inflammation can drive Pgp upregulation, we evaluated the ability of the new generation dual anti-inflammatory drug licofelone to promote spinal cord delivery of riluzole following SCI. We found that licofelone both reduced Pgp expression and enhanced riluzole bioavailability within the lesion site at 72 h post-SCI. This work highlights Pgp-mediated drug resistance as an important obstacle to therapeutic drug delivery for SCI, and suggests licofelone as a novel combinatorial treatment strategy to enhance therapeutic drug delivery to the injured spinal cord.

Galveston Brain Injury Conference 2010: clinical and experimental aspects of blast injury.

Blast injury is the most prevalent source of mortality and morbidity among combatants in Operations Iraqi and Enduring Freedom. Blast-induced neurotrauma (BINT) is a common cause of mortality, and even mild BINT may be associated with chronic cognitive and emotional deficits. In addition to military personnel, the increasing use of explosives by terrorists has resulted in growing numbers of blast injuries in civilian populations. Since the medical and rehabilitative communities are likely to be faced with increasing numbers of patients suffering from blast injury, the 2010 Galveston Brain Injury Conference focused on topics related to the diagnosis, treatment, and mechanisms of BINT. Although past military actions have resulted in large numbers of blast casualties, BINT is considered the signature injury of the conflicts in Iraq and Afghanistan. The attention focused on BINT has led to increased financial support for research on blast effects, contributing to the development of better experimental models of blast injury and a clearer understanding of the mechanisms of BINT. This more thorough understanding of blast injury mechanisms will result in novel and more effective therapeutic and rehabilitative strategies designed to reduce injury and facilitate recovery, thereby improving long-term outcomes in patients suffering from the devastating and often lasting effects of BINT. The following is a summary of the 2010 Galveston Brain Injury Conference, that included presentations related to the diagnosis and treatment of acute BINT, the evaluation of the long-term neuropsychological effects of BINT, summaries of current experimental models of BINT, and a debate about the relative importance of primary blast effects on the acute and long-term consequences of blast exposure.

Suprapubic bladder catheterization of male spinal-cord-injured Sprague-Dawley rats.

The rat spinal-cord-injury (SCI) model is widely used to study the pathologic mechanisms that contribute to sensory and motor dysfunction in humans. This model is thought to mimic many of the negative outcomes experienced by humans after spinal contusion injury. We theorized that manual bladder expression contributed to the kidney and bladder lesions reported in previous studies using the rat SCI model. In the present study, rats were surgically implanted with bladder catheters after spinal contusion injury to provide continuous drainage of urine. After 72 h, the rats were euthanized and their kidneys and bladders examined histologically. BUN, serum creatinine, and urine protein were compared at 0 and 72 h after surgery. Kidney and bladder lesions were similar in SCI rats with and without implanted bladder catheters. BUN at 72 h was higher than baseline values in both groups, whereas serum creatinine was higher at 72 h compared with baseline values only in the catheterized rats. These findings indicate that suprapubic bladder catheterization does not reduce hydronephrosis in SCI rats and that the standard of care for bladder evacuation should continue to be manual expression of urine.

Histopathological and behavioral effects of immediate and delayed hemorrhagic shock after mild traumatic brain injury in rats.

The purpose of this study was to investigate the increased susceptibility of the brain, after a controlled mild cortical impact injury, to a secondary ischemic insult. The effects of the duration and the timing of the secondary insult after the initial cortical injury were studied. Rats anesthetized with isoflurane underwent a 3 m/sec, 2.5-mm deformation cortical impact injury followed by hypotension to 40 mm Hg induced by withdrawing blood from a femoral vein. The duration of hypotension was varied from 40 to 60 min. The timing of 60 min of hypotension was varied from immediately post-injury to 7 days after the injury. Outcome was assessed by behavioral tasks and histological examination at 2 weeks post-injury. A separate group of animals underwent measurement of the acute physiology including mean blood pressure (MAP), intracranial pressure (ICP), and cerebral blood flow (CBF) using a laser Doppler technique. Increasing durations of hypotension resulted in marked expansion of the contusion, from 6.5±1.8 mm³ with sham hypotension to 27.1±3.9 mm³ with 60 min of hypotension. This worsening of the contusion was found only when then hypotension occurred immediately after injury or at 1 h after injury. CA3 neuron loss followed a similar pattern, but the injury group differences were not significant. Motor tasks, including beam balance and beam walking, were significantly worse following 50 and 60 min of hypotension. Performance on the Morris water maze task was also significantly related to the injury group. Studies of the acute cerebral hemodynamics demonstrated that CBF was significantly more impaired during hypotension in the animals that underwent the mild TBI compared to those that underwent sham TBI. The perfusion deficit was worst at the impact site, but also significant in the pericontusional brain. With 50 and 60 min of hypotension, CBF did not recover following resuscitation at the impact site, and recovered only transiently in the pericontusional brain. These results demonstrate that mild TBI, like more severe levels of TBI, can impair the brain's ability to maintain CBF during a period of hypotension, and result in a worse outcome.

Spinal cord injury triggers an intrinsic growth-promoting state in nociceptors.

Although most investigations of the mechanisms underlying chronic pain after spinal cord injury (SCI) have examined the central nervous system (CNS), recent studies have shown that nociceptive primary afferent neurons display persistent hyperexcitability and spontaneous activity in their peripheral branches and somata in dorsal root ganglia (DRG) after SCI. This suggests that SCI-induced alterations of primary nociceptors contribute to central sensitization and chronic pain after SCI. Does SCI also promote growth of these neurons' fibers, as has been suggested in some reports? The present study tests the hypothesis that SCI induces an intrinsic growth-promoting state in DRG neurons. This was tested by dissociating DRG neurons 3 days or 1 month after spinal contusion injury at thoracic level T10 and measuring neuritic growth 1 day later. Neurons cultured 3 days after SCI exhibited longer neurites without increases in branching ("elongating growth"), compared to neurons from sham-treated or untreated (naïve) rats. Robust promotion of elongating growth was found in small and medium-sized neurons (but not large neurons) from lumbar (L3-L5) and thoracic ganglia immediately above (T9) and below (T10-T11) the contusion site, but not from cervical DRG. Elongating growth was also found in neurons immunoreactive to calcitonin gene-related peptide (CGRP), suggesting that some of the neurons exhibiting enhanced neuritic growth were nociceptors. The same measurements made on neurons dissociated 1 month after SCI revealed no evidence of elongating growth, although evidence for accelerated initiation of neurite outgrowth was found. Under certain conditions this transient growth-promoting state in nociceptors might be important for the development of chronic pain and hyperreflexia after SCI.

Neuroprotection with an erythropoietin mimetic peptide (pHBSP) in a model of mild traumatic brain injury complicated by hemorrhagic shock.

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide, designed to interact with a novel cell surface receptor, composed of the classical erythropoietin (EPO) receptor disulfide linked to the beta common receptor. pHBSP has the cytoprotective effects of EPO without stimulating erythropoiesis. Effects on early cerebral hemodynamics and neurological outcome at 2 weeks post-injury were compared in a rat model of mild cortical impact injury (3m/sec, 2.5 mm deformation) followed by 50 min of hemorrhagic hypotension (MAP 40 mm Hg for 50 min). Rats were randomly assigned to receive 5000 U/kg of EPO, 30 µg/kg of pHBSP, or an inactive substance every 12 h for 3 days, starting at the end of resuscitation from the hemorrhagic hypotension, which was 110 min post-injury. Both treatments reduced contusion volume at 2 weeks post-injury, from 20.8±2.8 mm(3) in the control groups to 7.7±2.0 mm(3) in the EPO-treated group and 5.9±1.5 mm(3) in the pHBSP-treated group (p=0.001). Both agents improved recovery of cerebral blood flow in the injured brain following resuscitation, and resulted in more rapid recovery of performance on beam balancing and beam walking tests. These studies suggest that pHBSP has neuroprotective effects similar to EPO in this model of combined brain injury and hypotension. pHBSP may be more useful in the clinical situation because there is less risk of thrombotic adverse effects.

Longitudinal evaluation of mouse hind limb bone loss after spinal cord injury using novel, in vivo, methodology.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse, aberrant neuronal signaling and hormonal changes. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies. Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis. An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p < 0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p < 0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI (9). Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.