Shyam Gajavelli

Biomarkers for the clinical differential diagnosis in traumatic brain injury-A systematic review

Rapid triage and decision‐making in the treatment of traumatic brain injury (TBI) present challenging dilemma in “resource poor” environments such as the battlefield and developing areas of the world. There is an urgent need for additional tools to guide treatment of TBI. The aim of this review is to establish the possible use of diagnostic TBI biomarkers in (1) identifying diffuse and focal brain injury and (2) assess their potential for determining outcome, intracranial pressure (ICP), and responses to therapy. At present, there is insufficient literature to support a role for diagnostic biomarkers in distinguishing focal and diffuse injury or for accurate determination of raised ICP. Presently, neurofilament (NF), S100β, glial fibrillary acidic protein (GFAP), and ubiquitin carboxyl terminal hydrolase‐L1 (UCH‐L1) seemed to have the best potential as diagnostic biomarkers for distinguishing focal and diffuse injury, whereas C‐tau, neuron‐specific enolase (NSE), S100β, GFAP, and spectrin breakdown products (SBDPs) appear to be candidates for ICP reflective biomarkers. With the combinations of different pathophysiology related to each biomarker, a multibiomarker analysis seems to be effective and would likely increase diagnostic accuracy. There is limited research focusing on the differential diagnostic properties of biomarkers in TBI. This fact warrants the need for greater efforts to innovate sensitive and reliable biomarkers. We advocate awareness and inclusion of the differentiation of injury type and ICP elevation in further studies with brain injury biomarkers.

Absence of Major Histocompatibility Complex Class I on Neural Stem Cells Does Not Permit Natural Killer Cell Killing and Prevents Recognition by Alloreactive Cytotoxic T Lymphocytes In Vitro

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day‐13 embryonic (E13) B6 (H‐2b) mice were explanted and cultured to expand NSCs. Analysis of P2‐P17–cultured cells using anti‐MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon‐gamma (rmIFNγ) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFNγNSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFNγ for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFNγ‐treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H‐2d) allospecific anti‐H‐2b CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC‐1 target cells, these effectors did not kill MHC‐deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T‐ and NK‐cell populations.

TrkC overexpression enhances survival and migration of neural stem cell transplants in the rat spinal cord.

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 trkC-overexpressing 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.

Acute Diagnostic Biomarkers for Spinal Cord Injury: Review of the Literature and Preliminary Research Report

OBJECTIVE Many efforts have been made to create new diagnostic technologies for use in the diagnosis of central nervous system injury. However, there is still no consensus for the use of biomarkers in clinical acute spinal cord injury (SCI). The aims of this review are (1) to evaluate the current status of neurochemical biomarkers and (2) to discuss their potential acute diagnostic role in SCI by reviewing the literature. METHODS PubMed (http://www.ncbi.nlm.nih.gov/pubmed) was searched up to 2012 to identify publications concerning diagnostic biomarkers in SCI. To support more knowledge, we also checked secondary references in the primarily retrieved literature. RESULTS Neurofilaments, cleaved-Tau, microtubule-associated protein 2, myelin basic protein, neuron-specific enolase, S100β, and glial fibrillary acidic protein were identified as structural protein biomarkers in SCI by this review process. We could not find reports relating ubiquitin C-terminal hydrolase-L1 and α-II spectrin breakdown products, which are widely researched in other central nervous system injuries. Therefore, we present our preliminary data relating to these two biomarkers. Some of biomarkers showed promising results for SCI diagnosis and outcome prediction; however, there were unresolved issues relating to accuracy and their accessibility. CONCLUSION Currently, there still are not many reports focused on diagnostic biomarkers in SCI. This fact warranted the need for greater efforts to innovate sensitive and reliable biomarkers for SCI.

BMP signaling initiates a neural crest differentiation program in embryonic rat CNS stem cells

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 p75NTR, 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.

Neuroprotective effect of preoperatively induced mild hypothermia as determined by biomarkers and histopathological estimation in a rat subdural hematoma decompression model.

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.

Evidence to support mitochondrial neuroprotection, in severe traumatic brain injury

Traumatic brain injury (TBI) is still the leading cause of disability in young adults worldwide. The major mechanisms – diffuse axonal injury, cerebral contusion, ischemic neurological damage, and intracranial hematomas have all been shown to be associated with mitochondrial dysfunction in some form. Mitochondrial dysfunction in TBI patients is an active area of research, and attempts to manipulate neuronal/astrocytic metabolism to improve outcomes have been met with limited translational success. Previously, several preclinical and clinical studies on TBI induced mitochondrial dysfunction have focused on opening of the mitochondrial permeability transition pore (PTP), consequent neurodegeneration and attempts to mitigate this degeneration with cyclosporine A (CsA) or analogous drugs, and have been unsuccessful. Recent insights into normal mitochondrial dynamics and into diseases such as inherited mitochondrial neuropathies, sepsis and organ failure could provide novel opportunities to develop mitochondria-based neuroprotective treatments that could improve severe TBI outcomes. This review summarizes those aspects of mitochondrial dysfunction underlying TBI pathology with special attention to models of penetrating traumatic brain injury, an epidemic in modern American society.

Intraspinal transplantation of GABAergic neural progenitors attenuates neuropathic pain in rats: A pharmacologic and neurophysiological evaluation

Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABA(A) receptor antagonist bicuculline (BIC), GABA(B) receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1-3week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals.

Sustained analgesic peptide secretion and cell labeling using a novel genetic modification

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-β (NGF-β) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of γ‐aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA‐releasing 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 achaete‐scute 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 lentivirus‐transduced NPCs were maintained in FGF‐2 or deprived of FGF‐2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA‐ and β‐III‐tubulin‐immunoreactive 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 population 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 β‐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.