Miami Project to Cure Paralysis

Although CNS axons have the capacity to regenerate after spinal cord injury when provided with a permissive substrate, the lack of appropriate synaptic target sites for regenerating fibers may limit restoration of spinal circuitry. Studies in our laboratory are focused on utilizing neural stem cells to provide new synaptic target sites for regenerating spinal axons following injury. As an initial step, rat neural precursor cells genetically engineered to overexpress the tyrosine kinase C (trkC) neurotrophin receptor were transplanted into the intact rat spinal cord to evaluate their survival and differentiation. Cells were either pretreated in vitro prior to transplantation with trkC ligand neurotrophin-3 (NT-3) to initiate differentiation or exposed to NT-3 in vivo following transplantation via gelfoam or Oxycel. Both treatments enhanced survival of trkCoverexpressing stem cells to nearly 100%, in comparison with approximately 30-50% when either NT-3 or trkC was omitted. In addition, increased migration of trkC-overexpressing cells throughout the spinal gray matter was noted, particularly following in vivo NT-3 exposure. The combined trkC expression and NT-3 treatment appeared to reduce astrocytic differentiation of transplanted neural precursors. Decreased cavitation and increased β-tubulin fibers were noted in the vicinity of transplanted cells, although the majority of transplanted cells appeared to remain in an undifferentiated state. These findings suggest that genetically engineered neural stem cells in combination with neurotrophin treatment may be a useful addition to strategies for repair of spinal neurocircuitry following injury.

Bone morphogenetic proteins (BMPs) have an important role in neuronal and astrocytic differentiation of embryonic and adult neural stem cells (NSCs). Here, we show that BMP6, BMP7, GDF5, and GDF6 instructively differentiate E12, E14, and E17 rat cortical NSCs into a variety of neural crest lineages. Clonal analysis shows that BMP7-treated NSCs develop mostly into smooth muscle and peripheral glia. We observed a rapid induction of premigratory neural crest markers like p75 NTR , and AP-2 alpha followed by Msx1, Msx2, and Slug, transcription factors that participate in neural crest development. These results suggest that NSCs cultured in vitro in the presence of FGF2 display expanded developmental potential.

Differentiation of stem cells depends on environmental cues. In this study, acutely dissociated or expanded cells derived from embryonic day 14 (E14) rat cerebral cortex were transplanted into the distal tibial nerve stump of adult Fischer rats to determine whether a peripheral nervous system (PNS) environment would influence cell differentiation. Acutely dissociated cells, which included neural precursors and postmitotic neurons, were transplanted immediately after harvest. Expanded cortical cells were transplanted after 8 days of culture with fibroblast growth factor-2 (FGF-2), a process that yields a population of neural stem cells and/or neural precursors. After 2 or 10 weeks in peripheral nerve, the majority of the transplanted cells was astrocytes, as judged from glial fibrillary acid protein (GFAP) expression. Only acutely dissociated transplants had cells that exhibited neuronal phenotypes. Those neurons present in transplants at 10 weeks stained positive for glutamate decarboxylase and did not reinnervate muscle. Maintenance of this cortical phenotype in peripheral nerve suggests that it is necessary to transplant cells with neural phenotypes appropriate for muscle to restore its function.

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of ␥-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABAreleasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaetescute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirustransduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA-and ␤-III-tubulinimmunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased popu-lation of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased ␤-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function. J. Comp. Neurol. 515:56 -71, 2009. , 2007). Most current therapeutic strategies for treating these disorders involve the systemic administration of drugs that compensate for the loss of inhibitory signaling, sometimes producing undesirable cognitive and behavioral side effects. Cell transplantation could potentially supplement deficient CNS cell populations and deliver focal, physiologically specific therapy. The transplantation of GABAergic cells has been shown to produce behavioral recovery in animal models of neurological disorders (Gernert et al.

Traumatic brain injury (TBI) is a major cause of death and disability in all age groups. Among TBI, penetrating traumatic brain
injuries (PTBI) have the worst prognosis and represent the leading cause of TBI-related morbidity and death. However, there are no
specific drugs/interventions due to unclear pathophysiology. To gain insights we looked at cerebral metabolism in a PTBI rat model:
penetrating ballistic-like brain injury (PBBI). Early after injury, regional cerebral oxygen tension and consumption significantly
decreased in the ipsilateral cortex in the PBBI group compared with the control group. At the same time point, glucose uptake was
significantly reduced globally in the PBBI group compared with the control group. Examination of Fluorojade B-stained brain
sections at 24 hours after PBBI revealed an incomplete overlap of metabolic impairment and neurodegeneration. As expected, the
injury core had the most severe metabolic impairment and highest neurodegeneration. However, in the peri-lesional area, despite
similar metabolic impairment, there was lesser neurodegeneration. Given our findings, the data suggest the presence of two
distinct zones of primary injury, of which only one recovers. We anticipate the peri-lesional area encompassing the PBBI ischemic
penumbra, could be salvaged by acute therapies.

Abstract
Background: The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare
problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic
methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting
multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our
lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the
mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated
the analgesic potential of these peptides when delivered via gene therapy.
Results: Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into
rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical
and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of
individually injected components were modest, but the combination of EMs and SHG produced robust and sustained
antinociception. The onset of the analgesic effects was observed between 1–5 weeks post-injection and sustained
without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of
SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection
of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence
of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of
these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in
order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in
these animals using the SHG-EMs combination vectors compared to the group with early intervention.
Conclusions: Findings from this study support the potential for direct gene therapy to provide a robust and sustained
alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a
naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance
development for long-term management of persistent pain.

BACKGROUND:

Neuromonitoring with microdialysis has the potential for early detection of metabolic derangements associated with TBI.
METHODS:

1,260 microdialysis samples from 12 TBI patients were analyzed for glucose, -lactate, pyruvate, lactate/pyruvate ratio (LPR), and lactate/glucose ratio (LGR). Analytes were correlated with the Glasgow Coma Scale (GCS) before surgery and with the Glasgow Outcome Scale (GOS) at the time of discharge. The patients were divided into two groups for GCS: 3-6 and 7-9, and for GOS 1-3 and 4-5. Chi-squared test was performed for correlations.
RESULTS:

Glucose, lactate levels, and LGR were high in TBI patients with GCS 3-6 (p < 0.0001). Pyruvate level was lower in patients with GCS 7-9 (p < 0.001). LPR was higher in patients with GCS 3-6 (p < 0.05). High glucose, lactate level (p < 0.001), and LPR (p < 0.01) was observed in patients with GOS 1-3. Pyruvate level was low in patients with GOS 1-3 (p < 0.001). LGR was higher in patient with better outcome (GOS 4-5).
CONCLUSION:

After craniotomy extracellular glucose and lactate were good "biomarkers" of cerebral damage in TBI patients. We consider that high extracellular lactate and low glucose is an indicator of severe neurological damage and poor outcome, because of impaired brain metabolism.

PMID:
    23564137
    [PubMed - indexed for MEDLINE]

OBJECT:

In patients who have sustained a traumatic brain injury (TBI), hypothermia therapy has not shown efficacy in multicenter clinical trials. Armed with the post hoc data from the latest clinical trial (National Acute Brain Injury Study: Hypothermia II), the authors hypothesized that hypothermia may be beneficial in an acute subdural hematoma (SDH) rat model by blunting the effects of ischemia/reperfusion injury. The major aim of this study was to test the efficacy of temperature management in reducing brain damage after acute SDH.
METHODS:

The rats were induced with acute SDH and placed into 1 of 4 groups: 1) normothermia group (37°C); 2) early hypothermia group, head and body temperature reduced to 33°C 30 minutes prior to craniotomy; 3) late hypothermia group, temperature lowered to 33°C 30 minutes after decompression; and 4) sham group, no acute SDH (only craniotomy with normothermia). To assess for neuronal and glial cell damage, the authors analyzed microdialysate concentrations of GFAP and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) by using a 100-kD probe. Fluoro-Jade B-positive neurons and injury volume with 2,3,5-triphenyltetrazolium chloride staining were also measured.
RESULTS:

In the early phase of reperfusion (30 minutes, 2.5 hours after decompression), extracellular UCH-L1 in the early hypothermia group was significantly lower than in the normothermia group (early, 4.9 ± 1.0 ng/dl; late, 35.2 ± 12.1 ng/dl; normothermia, 50.20 ± 28.3 ng/dl; sham, 3.1 ± 1.3 ng/dl; early vs normothermia, p < 0.01; sham vs normothermia, p < 0.01, analyzed using ANOVA followed by a post hoc Bonferroni test). In the late phase of reperfusion (> 2.5 hours after decompression), extracellular GFAP in the early hypothermia group was also lower than in the normothermia and late hypothermia groups (early, 5.5 ± 2.9 ng/dl; late, 7.4 ± 3.4 ng/dl; normothermia, 15.3 ± 8.4 ng/dl; sham, 3.3 ± 1.0 ng/dl; normothermia vs sham; p < 0.01). The number of Fluoro-Jade B-positive cells in the early hypothermia group was significantly smaller than that in the normothermia group (normothermia vs early: 774,588 ± 162,173 vs 180,903 ± 42,212, p < 0.05). Also, the injury area and volume were smaller in the early hypothermia group in which hypothermia was induced before craniotomy and cerebral reperfusion (early, 115.2 ± 15.4 mm(3); late, 344.7 ± 29.1 mm(3); normothermia, 311.2 ± 79.2 mm(3); p < 0.05).
CONCLUSIONS:

The data suggest that early, preoperatively induced hypothermia could mediate the reduction of neuronal and glial damage in the reperfusion phase of ischemia/reperfusion brain injury.

Neuropathic pain induced by spinal cord injury (SCI) is clinically challenging with inadequate long-term
treatment options. Partial pain relief offered by pharmacologic treatment is often counterbalanced by adverse
effects after prolonged use in chronic pain patients. Cell-based therapy for neuropathic pain using GABAergic
neuronal progenitor cells (NPC) has the potential to overcome untoward effects of systemic pharmacotherapy
while enhancing analgesic potency due to local activation of GABAergic signaling in the spinal cord. However,
multifactorial anomalies underlying chronic pain will likely require simultaneous targeting of multiple
mechanisms. Here we explore the analgesic potential of genetically modified rat embryonic GABAergic NPCs
releasing a peptidergic NMDA receptor antagonist, Serine1-histogranin (SHG), thus targeting both spinal
hyperexcitability and reduced inhibitory processes. Recombinant NPCs were designed using either lentiviral or
adeno-associated-viral vectors (AAV2/8) encoding single and multimeric (6 copies of SHG) cDNA. Intraspinal
injection of recombinant cells elicited enhanced analgesic effects compared to nonrecombinant NPCs in spinal
cord injury-induced pain in rats. Moreover, potent and sustained antinociception was achieved, even following a
5 weeks post-injury delay, using recombinant multimeric NPCs. Intrathecal injection of SHG antibody
attenuated analgesic effects of the recombinant grafts suggesting active participation of SHG in these
antinociceptive effects. Immunoblots and immunocytochemical assays indicated ongoing recombinant peptide
production and secretion in the grafted host spinal cords. These results support the potential for engineered
NPCs grafted into the spinal dorsal horn to alleviate chronic neuropathic pain

Chronic neuropathic pain represents clinically challenging state with poor response to current treatment options. Long-term management of chronic pain is often associated with the development of tolerance, addiction and other side effects, reducing the therapeutic value of treatment. Alternative strategies based on cell therapy and gene manipulation, balancing the inhibitory and excitatory events in the spinal cord, may provide sustained pain relief in the long term. Transplantation of GABAergic cells has been successfully used to enhance inhibition and to restore physiological spinal pain processing. However, since the underlying mechanism of chronic pain development involves changes in several pain signaling pathways, it is essential to develop an approach targeting several components of pain signaling. Recombinant cell therapy offers the possibility to deliver additional analgesic substances to the restricted area in the nervous system. The current study explores the analgesic potential of genetically modified rat embryonic GABAergic cells releasing a peptidergic NMDA receptor antagonist, Serine1-histogranin (SHG). Overactivation of glutamate NMDA receptors contributes to the hyperexcitability of spinal neurons observed in the chronic pain models. Our approach allows us to simultaneously target spinal hyperexcitability and reduced inhibitory processes. Transplantable cells were transduced by viral vectors encoding either 1 or 6 copies of SHG cDNAs. The analgesic potential of recombinant cells after their intraspinal transplantation was evaluated in a model of peripheral nerve injury. Enhanced reduction of hypersensitivity to thermal and mechanical stimuli was observed in animals treated by recombinant cells compared to the nonrecombinant group. The recombinant peptide was detected in the spinal tissue,suggesting its successful production by transplanted cells. Our results demonstrate feasibility of using recombinant cells releasing adjunct analgesic peptides in the therapy of neuropathic pain.

Traumatic brain injury (TBI) leads to a series of pathological events that can have profound influences on motor, sensory and cognitive functions. Conversely, TBI can also stimulate neural stem/progenitor cell proliferation leading to increased numbers of neuroblasts migrating outside their restrictive neurogenic zone to areas of damage in support of tissue integrity. Unfortunately, the factors that regulate migration are poorly understood. Here, we examine whether ephrinB3 functions to restrict neuroblasts from migrating outside the subventricular zone (SVZ) and rostral migratory stream (RMS). We have previously shown that ephrinB3 is expressed in tissues surrounding these regions, including the overlying corpus callosum (CC), and is reduced after controlled cortical impact (CCI) injury. Our current study takes advantage of ephrinB3 knockout mice to examine the influences of ephrinB3 on neuroblast migration into CC and cortex tissues after CCI injury. Both injury and/or ephrinB3 deficiency led to increased neuroblast numbers and enhanced migration outside the SVZ/RMS zones. Application of soluble ephrinB3-Fc molecules reduced neuroblast migration into the CC after injury and limited neuroblast chain migration in cultured SVZ explants. Our findings suggest that ephrinB3 expression in tissues surrounding neurogenic regions functions to restrict neuroblast migration outside the RMS by limiting chain migration.

Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies in penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread peri-lesional neurodegeneration, similar to that seen in humans following gunshot wound to head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Towards this, green fluorescent protein (GFP) labeled hNSCs (400,000 per animal) were transplanted in immunosuppressed Sprague Dawley (SD), Fisher, and athymic (ATN) PBBI rats one week after injury. Tacrolimus (3mg/kg two days prior to transplantation, then 1mg/kg/day), Methylprednisolone (10mg/kg on day of transplant,  1mg/kg/week thereafter), and Mycophenolate mofetil (30mg/kg/day) for seven days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8-weeks post transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16-weeks post-transplantation neither cell proliferation nor glial lineage markers expression was detected. Transplanted cell morphology was similar to neighboring host neurons and there was relatively little migration of cells from the peri-transplant site. By 16 weeks, GFP positive processes extended both rostro-caudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing internal capsule, extending ~13 mm caudally from transplantation site reaching into the brain stem. In a Morris water maze test at 8-weeks post transplantation, animals with transplants had shorter latency to platform compared to vehicle treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits of durable engraftment and neuronal differentiation. Thus, these results justify further studies to progress towards clinical translation of hNSC therapy for PTBI.

Penetrating traumatic brain injury (PTBI) is a significant cause of death and disability in the United States. Inflammasomes are one of the key regulators of the interleukin (IL)–1b mediated inflammatory responses after traumatic brain injury. However, the contribution of inflammasome signaling after PTBI has not been determined. In this study, adult male Sprague-Dawley rats were subjected to sham procedures or penetrating ballistic-like brain injury (PBBI) and sacrificed at various time-points. Tissues were assessed by immunoblot analysis for expression of IL-1b, IL-18, and components of the inflammasome: apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), caspase-1, X-linked inhibitor of apoptosis protein (XIAP), nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), and gasdermin-D (GSDMD). Specific cell types expressing inflammasome proteins also were evaluated immunohistochemically and assessed quantitatively. After PBBI, expression of IL-1b, IL-18, caspase-1, ASC, XIAP, and NLRP3 peaked around 48 h. Brain protein lysates from PTBI animals showed pyroptosome formation evidenced by ASC laddering, and also contained increased expression of GSDMD at 48 h after injury. ASC-positive immunoreactive neurons within the perilesional cortex were observed at 24 h. At 48 h, ASC expression was concentrated in morphologically activated cortical microglia. This expression of ASC in activated microglia persisted until 12 weeks following PBBI. This is the first report of inflammasome activation after PBBI. Our results demonstrate cell-specific patterns of inflammasome activation and pyroptosis predominantly in microglia, suggesting a sustained pro-inflammatory state following PBBI, thus offering a therapeutic target for this type of brain injury.