Pramod K. Dash

Enhanced neurogenesis in the rodent hippocampus following traumatic brain injury

Recent studies have shown that neurogenesis in the dentate gyrus of the rodent hippocampus continues throughout life. Several physiological and pathological conditions have been reported to alter the rate of progenitor cell division resulting in the increased production of mature granule neurons. Excitotoxic and mechanical lesions of the granule cell layer also stimulate the proliferation of precursor cells suggesting that the death of pre‐existing granule neurons may act as a trigger for enhanced neurogenesis. Hippocampal pyramidal neurons, and to a lesser extent granule neurons, have been reported to die as a result of traumatic brain injury in rodents. To determine if the proliferation of precursor cells is enhanced as a result of brain injury in rodents, newly divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). Traumatic brain injury increased the production of BrdU‐labeled cells in the dentate gyrus with a maximal rate observed at 3 days post‐injury. These cells, a proportion of which co‐localize with the immature neuronal marker TOAD‐64, implanted themselves into the granule cell layer where they accumulated over time. When examined 1 month post‐injury, the majority of BrdU‐labeled cells co‐labeled with the mature neuronal marker calbindin. These findings show that traumatic brain injury increases neurogenesis in the granule cell layer and suggests that these new cells may contribute to the function of the hippocampus. J. Neurosci. Res. 63:313–319, 2001.

Apoptotic morphology of dentate gyrus granule cells following experimental cortical impact injury in rats: possible role in spatial memory deficits

Loss of hippocampal neurons as a result of traumatic brain injury (TBI) is thought to contribute to the observed spatial memory deficits. Using a rodent model of experimental brain injury, we have examined the nature of hippocampal cell death following TBI. Light microscope examination of stained sections showed the presence of a large number of hyperchromatic and dystrophic neurons in the dentate gyrus of the hippocampus. These cells appeared to be undergoing nuclear condensation. Electron microscope examination demonstrated the presence of cell shrinkage, condensed chromatin, nuclear segmentation, and cytoplasmic vacuolization. Detection of a DNA ladder and in situ labeling (TUNEL) were also consistent with the process of apoptosis. However, in some dystrophic neurons these morphologies were also accompanied by the presence of swollen mitochondria and a lack of distinctive rough endoplasmic reticulum which are typically associated with necrosis. These findings show that cortical impact injury produces cell death in the hippocampus which has both apoptotic and necrotic features.

Sulforaphane reduces infarct volume following focal cerebral ischemia in rodents

Stroke is the third leading cause of death and disability in the United States. As several biochemical mechanisms have been proposed to contribute to stroke pathophysiology, treatments acting on multiple targets may be desirable. Sulforaphane (SUL), a naturally occurring isothiocyanate present in cruciferous vegetables, has been shown to induce the expression of multiple NF-E2-related factor-2 (Nrf2) responsive genes. In the present study, we demonstrate that systemically administered SUL can enter the brain as determined by increased mRNA and protein levels of the Nrf2-responsive gene heme oxygenase 1 (HO-1). Delayed administration (15 min) of a single dose of SUL significantly decreased cerebral infarct volume following focal ischemia, suggesting a potential therapeutic value for this compound.

Enhancing Expression of Nrf2-Driven Genes Protects the Blood–Brain Barrier after Brain Injury

The integrity of the blood–brain barrier (BBB) is critical for normal brain function, and its compromise contributes to the pathophysiology of a number of CNS diseases and injuries. Using a rodent model of brain injury, the present study examines the pathophysiology of BBB disruption. Western blot and immunohistochemical analyses indicate that brain injury causes a loss of capillary endothelial cells and tight junction proteins, two critical components of the BBB. Activation of the transcription factor NF-E2-related factor-2 (Nrf2) by sulforaphane, a naturally occurring compound present in high levels in cruciferous vegetables, significantly increased the expression of endogenous cytoprotective genes in brain tissue and microvessels as indicated by real-time PCR analysis. Postinjury administration of sulforaphane reduced the loss of endothelial cell markers and tight junction proteins and preserved BBB function. These protective effects were dependent on the activity of Nrf2. Injured rats pretreated with decoy oligonucleotides containing the binding site of Nrf2, and mice lacking the nrf2 gene, did not benefit from sulforaphane administration. These findings indicate a potential therapeutic usefulness for Nrf2-activating molecules to improve the function of the neurovascular unit after injury.

Delayed, selective neuronal death following experimental cortical impact injury in rats: possible role in memory deficits

Clinical and experimental studies show that loss of neurons in the hippocampus and/or the entorhinal cortex can impede formation and storage of spatial memory. Using a controlled cortical impact model of traumatic brain injury (TBI) in rats, we have examined the temporal and spatial pattern of neuronal death using silver impregnation and cresyl violet staining. Dystrophic neurons can be detected in the dentate gyrus, and the CA1 and CA3 subfields of the hippocampus for up to 2 weeks following injury. These dystrophic cells appeared shrunken and possessed features of apoptosis. Areas containing the dystrophic cells suffer substantial cell loss as demonstrated by thinning of the neuronal layers. Dystrophic cells are also found in the amygdala, entorhinal and piriform cortices, thalamic and hypothalamic regions, and surrounding the contusion site. The loss of these cells may contribute to the memory deficits observed following TBI.

A role for prefrontal cortex in memory storage for trace fear conditioning

The prefrontal cortex has been shown to participate in the association of events separated by time. However, it is not known whether the prefrontal cortex stores the memory for these relationships. Trace conditioning is a form of classical conditioning in which a time gap separates the conditioned stimulus (CS) from the unconditioned stimulus (US), the association of which has been shown to depend on prefrontal activity. Here we demonstrate that inhibition of extracellular signal-regulated kinase (Erk) cascade (a biochemical pathway involved in long-term memory storage) in the rat medial prefrontal cortex did not interfere with memory encoding for trace fear conditioning but impaired memory retention. In addition, animals displayed impaired memory for the irrelevancy of the training context. Hippocampal Erk phosphorylation was found to have a later time course than prefrontal Erk phosphorylation after trace fear conditioning, indicating a direct role for the prefrontal cortex in associative memory storage for temporally separated events as well as in memory storage of relevancy.

High-density microarray analysis of hippocampal gene expression following experimental brain injury.

Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. Traumatic brain injury (TBI) often causes spatial memory deficits, which are thought to arise from the death as well as the dysfunction of hippocampal neurons. Cell death and dysfunction are commonly associated with and often caused by altered expression of specific genes. The identification of the genes involved in these processes, as well as those participating in postinjury cellular repair and plasticity, is important for the development of mechanism‐based therapies. To monitor the expression levels of a large number of genes and to identify genes not previously implicated in TBI pathophysiology, a high‐density oligonucleotide array containing 8,800 genes was interrogated. RNA samples were prepared from ipsilateral hippocampi 3 hr and 24 hr following lateral cortical impact injury and compared to samples from sham‐operated controls. Cluster analysis was employed using statistical algorithms to arrange the genes according to similarity in patterns of expression. The study indicates that the genomic response to TBI is complex, affecting approximately 6% (at the time points examined) of the total number of genes examined. The identity of the genes revealed that TBI affects many aspects of cell physiology, including oxidative stress, metabolism, inflammation, structural changes, and cellular signaling. The analysis revealed genes whose expression levels have been reported to be altered in response to injury as well as several genes not previously implicated in TBI pathophysiology.

Human Traumatic Brain Injury Alters Plasma microRNA Levels

Circulating microRNAs (miRNAs) present in the serum/plasma are characteristically altered in many pathological conditions, and have been employed as diagnostic markers for specific diseases. We examined if plasma miRNA levels are altered in patients with traumatic brain injury (TBI) relative to matched healthy volunteers, and explored their potential for use as diagnostic TBI biomarkers. The plasma miRNA profiles from severe TBI patients (Glasgow Coma Scale [GCS] score ≤8) and age-, gender-, and race-matched healthy volunteers were compared by microarray analysis. Of the 108 miRNAs identified in healthy volunteer plasma, 52 were altered after severe TBI, including 33 with decreased and 19 with increased relative abundance. An additional 8 miRNAs were detected only in the TBI plasma. We used quantitative RT-PCR to determine if plasma miRNAs could identify TBI patients within the first 24 h post-injury. Receiver operating characteristic curve analysis indicated that miR-16, miR-92a, and miR-765 were good markers of severe TBI (0.89, 0.82, and 0.86 AUC values, respectively). Multiple logistic regression analysis revealed that combining these miRNAs markedly increased diagnostic accuracy (100% specificity and 100% sensitivity), compared to either healthy volunteers or orthopedic injury patients. In mild TBI patients (GCS score > 12), miR-765 levels were unchanged, while the plasma levels of miR-92a and miR-16 were significantly increased within the first 24 h of injury compared to healthy volunteers, and had AUC values of 0.78 and 0.82, respectively. Our results demonstrate that circulating miRNA levels are altered after TBI, providing a rich new source of potential molecular biomarkers. Plasma-derived miRNA biomarkers, used in combination with established clinical practices such as imaging, neurocognitive, and motor examinations, have the potential to improve TBI patient classification and possibly management.

Transcription Factor Nrf2 Protects the Brain From Damage Produced by Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) remains a major medical problem for which there is no effective treatment. Oxidative and cytotoxic damage plays an important role in ICH pathogenesis and may represent a target for treatment of ICH. Recent studies have suggested that nuclear factor–erythroid 2–related factor 2 (Nrf2), a pleiotropic transcription factor, may play a key role in protecting cells from cytotoxic/oxidative damage. This study evaluated the role of Nrf2 in protecting the brain from ICH-mediated damage. Methods— Sprague-Dawley rats and Nrf2-deficient or control mice received intracerebral injection of autologous blood to mimic ICH. Sulforaphane was used to activate Nrf2. Oxidative stress, the presence of myeloperoxidase-positive cells (neutrophils) in ICH-affected brains, and behavioral dysfunction were assessed to determine the extent of ICH-mediated damage. Results— Sulforaphane activated Nrf2 in ICH-affected brain tissue and reduced neutrophil count, oxidative damage, and behavioral deficits caused by ICH. Nrf2-deficient mice demonstrated more severe neurologic deficits after ICH and did not benefit from the protective effect of sulforaphane. Conclusions— Nrf2 may represent a strategic target for ICH therapies.

Valproate Administered after Traumatic Brain Injury Provides Neuroprotection and Improves Cognitive Function in Rats

Background Traumatic brain injury (TBI) initiates a complex series of neurochemical and signaling changes that lead to pathological events including neuronal hyperactivity, excessive glutamate release, inflammation, increased blood-brain barrier (BBB) permeability and cerebral edema, altered gene expression, and neuronal dysfunction. It is believed that a drug combination, or a single drug acting on multiple targets, may be an effective strategy to treat TBI. Valproate, a widely used antiepileptic drug, has a number of targets including GABA transaminase, voltage-gated sodium channels, glycogen synthase kinase (GSK)-3, and histone deacetylases (HDACs), and therefore may attenuate a number of TBI-associated pathologies. Methodology/Principal Findings Using a rodent model of TBI, we tested if post-injury administration of valproate can decrease BBB permeability, reduce neural damage and improve cognitive outcome. Dose-response studies revealed that systemic administration of 400 mg/kg (i.p.), but not 15, 30, 60 or 100 mg/kg, increases histone H3 and H4 acetylation, and reduces GSK-3 activity, in the hippocampus. Thirty min post-injury administration of 400 mg/kg valproate improved BBB integrity as indicated by a reduction in Evans Blue dye extravasation. Consistent with its dose response to inhibit GSK-3 and HDACs, valproate at 400 mg/kg, but not 100 mg/kg, reduced TBI-associated hippocampal dendritic damage, lessened cortical contusion volume, and improved motor function and spatial memory. These behavioral improvements were not observed when SAHA (suberoylanilide hydroxamic acid), a selective HDAC inhibitor, was administered. Conclusion/Significance Our findings indicate that valproate given soon after TBI can be neuroprotective. As clinically proven interventions that can be used to minimize the damage following TBI are not currently available, the findings from this report support the further testing of valproate as an acute therapeutic strategy.