Kian Chye Ng

Adult bone marrow cells differentiate into neural phenotypes and improve functional recovery in rats following traumatic brain injury

This study aims to investigate the therapeutic potential of adult bone marrow stromal cells (BMSCs). Exposed to a cocktail of induction medium, some BMSCs could differentiate into cell types with phenotypes of neural lineages in vitro. These cells expressed neural markers nestin, GFAP, 68-kDa neurofilament and beta-tubulin III as detected by immunohistochemistry and RT-PCR. Fluorescence-labeled cells were injected intravenously at 72 h after traumatic brain injury. Transplanted cells survived and migrated to the ipsilateral cerebral cortex at different time points after injection. They were immunopositive for neuronal marker MAP-2, oligodendrocyte marker CNPase, astrocytic maker GFAP or microglial marker OX-42 in vivo. In rats receiving BMSC transplants, there were significant improvements in motor and neurological functions when compared with the control groups. Hence, the therapeutic potential of BMSCs for traumatic brain injury is further amplified.

Effect of Blast Exposure on the Brain Structure and Cognition in Macaca fascicularis

Blast injury to the brain is one of the major causes of death and can also significantly affect cognition and physical and psychological skills in survivors of blast. The complex mechanisms via which blast injury causes impairment of cognition and other symptoms are poorly understood. In this study, we investigated the effects of varying degrees of primary blast overpressure (BOP; 80 and 200 kPa) on the pathophysiological and magnetic resonance imaging (MRI) changes and neurocognitive performance as assessed by the monkey Cambridge Neuropsychological Test Automated Battery (mCANTAB) in non-human primates (NHP). The study aimed to examine the effects of neurobehavioral and histopathological changes in NHP. MRI and histopathology revealed ultrastructural changes in the brain, notably in the Purkinje neurons in the cerebellum and pyramidal neurons in the hippocampus, which were most vulnerable to the blast. The results correlated well with the behavioral changes and changes in motor coordination and working memory of the affected monkeys. In addition, there was white matter damage affecting myelinated axons, astrocytic hypertrophy, and increased aquaporin-4 (AQP-4) expression in astrocytes, suggesting cerebral edema. Increased apoptosis appeared to involve astrocytes and oligodendrocytes in the animals following blast exposure. The small sample size could have contributed to the non-significant outcome in cognitive performance post-blast and limited quantitative analyses. Nevertheless, the study has provided initial descriptive changes for establishing a primary BOP threshold for brain injury to serve as a useful platform for future investigations that aim to estimate brain injury potential and set safe limits of exposure.

Neuroprotection by aminoguanidine after lateral fluid-percussive brain injury in rats: a combined magnetic resonance imaging, histopathologic and functional study.

The present study examined the effects of a selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on neuronal cell survival and post-traumatic recovery in rats following a lateral fluid percussive brain injury. Daily treatment of AG at the dosage of 100 mg/kg or normal saline was given intraperitoneally into rats starting 2 h before or 30 min after brain injury. Treatment with AG significantly reduced lesion volumes in the brains of rats after injury, as evaluated by high-resolution magnetic resonance imaging (MRI). Immunohistochemical analysis showed a marked induction of iNOS expression in brain macrophages ipsilateral to the injury. Apoptotic neurons were observed in the ipsilateral cerebral cortex by in situ terminal transferase d-UTP nick-end labelling (TUNEL) and caspase-3 immunohistochemistry. In rats receiving prophylactic or post-injury treatment of AG, the number of degenerating neurons was markedly reduced in the cerebrum compared to those receiving saline injection. The location and extent of these pathologic changes correlated with MRI findings. Neurobehavioral studies showed that rotametric performance, grip-strength score, total and ambulatory locomotor responses and acoustic startle response were reduced in rats subjected to the injury but were significantly improved in AG-treated rats. It is suggested that inhibition of iNOS by AG may represent a potential therapeutic strategy for the treatment of traumatic brain injury.

Low Level Primary Blast Injury in Rodent Brain

The incidence of blast attacks and resulting traumatic brain injuries has been on the rise in recent years. Primary blast is one of the mechanisms in which the blast wave can cause injury to the brain. The aim of this study was to investigate the effects of a single sub-lethal blast over pressure (BOP) exposure of either 48.9 kPa (7.1 psi) or 77.3 kPa (11.3 psi) to rodents in an open-field setting. Brain tissue from these rats was harvested for microarray and histopathological analyses. Gross histopathology of the brains showed that cortical neurons were “darkened” and shrunken with narrowed vasculature in the cerebral cortex day 1 after blast with signs of recovery at day 4 and day 7 after blast. TUNEL-positive cells were predominant in the white matter of the brain at day 1 after blast and double-labeling of brain tissue showed that these DNA-damaged cells were both oligodendrocytes and astrocytes but were mainly not apoptotic due to the low caspase-3 immunopositivity. There was also an increase in amyloid precursor protein immunoreactive cells in the white matter which suggests acute axonal damage. In contrast, Iba-1 staining for macrophages or microglia was not different from control post-blast. Blast exposure altered the expression of over 5786 genes in the brain which occurred mostly at day 1 and day 4 post-blast. These genes were narrowed down to 10 overlapping genes after time-course evaluation and functional analyses. These genes pointed toward signs of repair at day 4 and day 7 post-blast. Our findings suggest that the BOP levels in the study resulted in mild cellular injury to the brain as evidenced by acute neuronal, cerebrovascular, and white matter perturbations that showed signs of resolution. It is unclear whether these perturbations exist at a milder level or normalize completely and will need more investigation. Specific changes in gene expression may be further evaluated to understand the mechanism of blast-induced neurotrauma.

A Model for Mild Traumatic Brain Injury that Induces Limited Transient Memory Impairment and Increased Levels of Axon Related Serum Biomarkers

Mild traumatic brain injury (mTBI) is one of the most common neuronal insults and can lead to long-term disabilities. mTBI occurs when the head is exposed to a rapid acceleration-deceleration movement triggering axonal injuries. Our limited understanding of the underlying pathological changes makes it difficult to predict the outcome of mTBI. In this study we used a scalable rat model for rotational acceleration TBI, previously characterized for the threshold of axonal pathology. We have analyzed whether a TBI just above the defined threshold would induce any detectable behavioral changes and/or changes in serum biomarkers. The effect of injury on sensory motor functions, memory and anxiety were assessed by beam walking, radial arms maze and elevated plus maze at 3–7 days following TBI. The only behavioral deficits found were transient impairments in working and reference memory. Blood serum was analyzed at 1, 3, and 14 days after injury for changes in selected protein biomarkers. Serum levels of neurofilament heavy chain and Tau, as well as S100B and myelin basic protein showed significant increases in the injured animals at all time points. No signs of macroscopic injuries such as intracerebral hematomas or contusions were found. Amyloid precursor protein immunostaining indicated axonal injuries at all time points analyzed. In summary, this model mimics some of the key symptoms of mTBI, such as transient memory impairment, which is paralleled by an increase in serum biomarkers. Our findings suggest that serum biomarkers may be used to detect mTBI. The model provides a suitable foundation for further investigation of the underlying pathology of mTBI.

Characterization of a Novel Rat Model of Penetrating Traumatic Brain Injury

A penetrating traumatic brain injury (pTBI) occurs when an object impacts the head with sufficient force to penetrate the skin, skull, and meninges, and inflict injury directly to the brain parenchyma. This type of injury has been notoriously difficult to model in small laboratory animals such as rats or mice. To this end, we have established a novel non-fatal model for pTBI based on a modified air rifle that accelerates a pellet, which in turn impacts a small probe that then causes the injury to the experimental animals brain. In the present study, we have focused on the acute phase and characterized the tissue destruction, including increasing cavity formation, white matter degeneration, hemorrhage, edema, and gliosis. We also used a battery of behavioral models to examine the neurological outcome, with the most noteworthy finding being impairment of reference memory function. In conclusion, we have described a number of events taking place after pTBI in our model. We expect this model will prove useful in our efforts to unravel the biological events underlying injury and regeneration after pTBI and possibly serve as a useful animal model in the development of novel therapeutic and diagnostic approaches.

Nitric oxide induces macrophage apoptosis following traumatic brain injury in rats

This study examined the apoptotic mechanisms of macrophages following a lateral fluid percussive brain injury. A marked induction of inducible NO synthase (iNOS) immunoexpression was observed in brain macrophages in the subarachnoid space and lateral ventricles ipsilateral to the injury. Numerous apoptotic macrophages occurred in the same region 7 days after the injury as shown by in situ terminal transferase d-UTP nick-end labeling (TUNEL) and caspase-3 immunohistochemistry. Double immunofluorescence staining showed that only a small number of TUNEL positive cells were iNOS positive; many TUNEL positive cells, however, were observed in the vicinity of iNOS positive cells. Administration of aminoguanidine resulted in a marked reduction of apoptotic cells in the lesioned area suggesting that overproduction of NO is linked to diminution of brain macrophages by apoptosis.

Primary blast injury-induced lesions in the retina of adult rats

AbstractBackgroundThe effect of primary blast exposure on the brain is widely reported but its effects on the eye remains unclear. Here, we aim to examine the effects of primary blast exposure on the retina.MethodsAdult male Sprague–Dawley rats were exposed to primary blast high and low injury and sacrificed at 24 h, 72 h, and 2 weeks post injury. The retina was subjected to western analysis for vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4), glutamine synthethase (GS), inducible nitric oxide synthase (NOS), endothelial NOS, neuronal NOS and nestin expression; ELISA analysis for cytokines and chemokines; and immunofluorescence for glial fibrillary acidic protein (GFAP)/VEGF, GFAP/AQP4, GFAP/nestin, GS/AQP4, lectin/iNOS, and TUNEL.ResultsThe retina showed a blast severity-dependent increase in VEGF, iNOS, eNOS, nNOS, and nestin expression with corresponding increases in inflammatory cytokines and chemokines. There was also increased AQP4 expression and retinal thickness after primary blast exposure that was severity-dependent. Finally, a significant increase in TUNEL+ and Caspase-3+ cells was observed. These changes were observed at 24 h post-injury and sustained up to 2 weeks post injury.ConclusionsPrimary blast resulted in severity-dependent pathological changes in the retina, manifested by the increased expression of a variety of proteins involved in inflammation, edema, and apoptosis. These changes were observed immediately after blast exposure and sustained up to 2 weeks suggesting acute and chronic injury mechanisms. These changes were most obvious in the astrocytes and Müller cells and suggest important roles for these cells in retina pathophysiology after blast.

Preservation of neurological functions by nitric oxide synthase inhibitors following hemorrhagic shock

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated in the pathophysiology of hemorrhagic shock. Our aim was to study the effect of iNOS inhibitors, L-canavanine (50mg/kg) and N(G)-nitro- L-arginine methyl (L-NAME, 10mg/kg) and a resuscitation fluid, lactated Ringers solution (3 times amount of blood lost), on survivability and neurological functions in rodents subjected to hemorrhagic shock. L-canavanine-treated rats had significantly higher survival rates (75%) compared to L-NAME-treated rats (44%) and lactated Ringers solution-treated rats (40%), 72 h following hemorrhagic shock. A marked increase in the neurological performance was observed in L-canavanine-treated rats over the three-day period. Histological examinations also showed a reduction of degenerating neurons in L-canavanine-treated rats when compared to L-NAME-, lactated Ringers solution- or un-treated rats. Mean arterial blood pressure (MABP), nitrate/nitrite level, glutamic oxalacetic transaminase (GOT) level, and blood gases were also significantly improved in L-canavanine-treated rats when compared to those of L-NAME-, lactated Ringers solution- or un-treated rats. In conclusion, L-canavanine-treated rats were able to improve survivability, attenuate organ damage, and improve neurological outcome when compared to other treatment groups. It is therefore suggest that L-canavanine may be beneficial as a potentially useful therapeutic agent in treating neurological deficit as a result of hemorrhagic shock.

Multi-echo susceptibility-weighted imaging and histology of open-field blast-induced traumatic brain injury in a rat model.

Blast‐induced traumatic brain injury is on the rise, predominantly as a result of the use of improvised explosive devices, resulting in undesirable neuropsychological dysfunctions, as demonstrated in both animals and humans. This study investigated the effect of open‐field blast injury on the rat brain using multi‐echo, susceptibility‐weighted imaging (SWI). Multi‐echo SWI provided phase maps with better signal‐to‐noise ratio (SNR) and contrast‐to‐noise ratio (CNR), making it a sensitive technique for brain injury. Male Sprague‐Dawley rats were subjected to a survivable blast of 180 kPa. The visibility of blood vessels of varying sizes improved with multi‐echo SWI. Reduced signal intensity from major vessels post‐blast indicates increased deoxyhaemoglobin. Relative cerebral blood flow was computed from filtered phase SWI images using inferred changes in oxygen saturation from major blood vessels. Cerebral blood flow decreased significantly at day 3 and day 5 post‐blast compared with that pre‐blast. This was substantiated by the upregulation of β‐amyloid precursor protein (β‐APP), a marker of ischaemia, in the neuronal perikaya of the cerebral cortex, as observed by immunofluorescence, and in the cortical tissue by western blot analysis. Our findings indicate the presence of brain ischaemia in post‐blast acute phase of injury with possible recovery subsequently. Our results from cerebrovascular imaging, histology and staining provide an insight into the ischaemic state of the brain post‐blast and may be useful for prognosis and outcome. Copyright