Laura B. Ngwenya

Light and Electron Microscopic Immunohistochemical Detection of Bromodeoxyuridine-labeled Cells in the Brain: Different Fixation and Processing Protocols

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.

Maturational Sequence of Newly Generated Neurons in the Dentate Gyrus of the Young Adult Rhesus Monkey

The generation of new neurons in the hippocampal dentate gyrus of adult mammals has been characterized in rodents, but the details of this process have not been described in the primate. Eleven young adult rhesus monkeys were given an injection of the DNA synthesis phase marker bromodeoxyuridine (BrdU) and killed at varying survival intervals (2 hours to 98 days). The immature neuronal marker TUC‐4 (TOAD/Ulip/CRMP‐4) was used to define three stages of morphological maturation. Stage I neurons had small somata and lacked dendrites. Stage II neurons had larger somata and short dendrites. Stage III neurons were similar in size to mature granule cells and had branching dendrites that extended into the molecular layer. Examination of TUC‐4‐positive immature neurons colabeled with BrdU indicated that stage I neurons first appeared 2 days after BrdU injection, stage II neurons at 14 days, and stage III neurons at 35 days. Electron microscopy of TUC‐4‐labeled cells showed that stage I cells had ultrastructural features of immature neurons, whereas stage III neurons were similar to mature granule cells and formed synapses in the molecular layer. This suggests that stage III neurons could potentially integrate into the circuitry of the dentate gyrus. This study shows that the maturational sequence for new neurons in the adult monkey is similar to that of the adult rodent; however, maturation takes a minimum of 5 weeks in the monkey, which is substantially longer than what has been reported in rodents. J. Comp. Neurol. 498:204–216, 2006.

Age-related changes in dentate gyrus cell numbers, neurogenesis, and associations with cognitive impairments in the rhesus monkey

The generation of new neurons in the adult mammalian brain is well-established for the hippocampal dentate gyrus (DG). However, the role of neurogenesis in hippocampal function and cognition, how it changes in aging, and the mechanisms underlying this are yet to be elucidated in the monkey brain. To address this, we investigated adult neurogenesis in the DG of 42 rhesus monkeys (39 cognitively tested) ranging in age from young adult to the elderly. We report here that there is an age-related decline in proliferation and a delayed development of adult neuronal phenotype. Additionally, we show that many of the new neurons survive throughout the lifetime of the animal and may contribute to a modest increase in total neuron number in the granule cell layer of the DG over the adult life span. Lastly, we find that measures of decreased adult neurogenesis are only modestly predictive of age-related cognitive impairment.

Evaluation of Long-Term Cryostorage of Brain Tissue Sections for Quantitative Histochemistry

Storage of tissue sections for long periods allows multiple samples, acquired over months or years, to be processed together, in the same reagents, for quantitative histochemical studies. Protocols for freezer storage of free-floating frozen sections using sucrose with different additives have been reported and assert that storage has no effect on histochemistry, but no quantitative support has been provided. The present study analyzed the efficacy of long-term storage of brain tissue sections at −80C in buffered 15% glycerol. To determine whether histochemical reactivity is affected, we analyzed 11 datasets from 80 monkey brains that had sections stored for up to 10 years. For processing, sections from multiple cases were removed from storage, thawed, and batch-processed at the same time for different histochemical measures, including IHC for neuronal nuclear antigen, parvalbumin, orexin-A, doublecortin, bromodeoxyuridine, the pro-form of brain-derived neurotrophic factor, and damaged myelin basic protein as well as a histochemical assay for hyaluronic acid. Results were quantified using stereology, optical densitometry, fluorescence intensity, or percent area stained. Multiple regression analyses controlling for age and sex demonstrated the general stability of these antigens for up to a decade when stored in 15% glycerol at −80C.

Selective Serotonin Reuptake Inhibitors for Treating Neurocognitive and Neuropsychiatric Disorders Following Traumatic Brain Injury: An Evaluation of Current Evidence

The prevalence of neuropsychiatric disorders following traumatic brain injury (TBI) is 20%–50%, and disorders of mood and cognition may remain even after recovery of neurologic function is achieved. Selective serotonin reuptake inhibitors (SSRI) block the reuptake of serotonin in presynaptic cells to lead to increased serotonergic activity in the synaptic cleft, constituting first-line treatment for a variety of neurocognitive and neuropsychiatric disorders. This review investigates the utility of SSRIs in treating post-TBI disorders. In total, 37 unique reports were consolidated from the Cochrane Central Register and PubMed (eight randomized-controlled trials (RCTs), nine open-label studies, 11 case reports, nine review articles). SSRIs are associated with improvement of depressive but not cognitive symptoms. Pooled analysis using the Hamilton Depression Rating Scale demonstrate a significant mean decrease of depression severity following sertraline compared to placebo—a result supported by several other RCTs with similar endpoints. Evidence from smaller studies demonstrates mood improvement following SSRI administration with absent or negative effects on cognitive and functional recovery. Notably, studies on SSRI treatment effects for post-traumatic stress disorder after TBI remain absent, and this represents an important direction of future research. Furthermore, placebo-controlled studies with extended follow-up periods and concurrent biomarker, neuroimaging and behavioral data are necessary to delineate the attributable pharmacological effects of SSRIs in the TBI population.

Brain Tissue Oxygen Monitoring and the Intersection of Brain and Lung: A Comprehensive Review

Traumatic brain injury is a problem that affects millions of Americans yearly and for which there is no definitive treatment that improves outcome. Continuous brain tissue oxygen (PbtO2) monitoring is a complement to traditional brain monitoring techniques, such as intracranial pressure and cerebral perfusion pressure. PbtO2 monitoring has not yet become a clinical standard of care, due to several unresolved questions. In this review, we discuss the rationale and technology of PbtO2 monitoring. We review the literature, both historic and current, and show that continuous PbtO2 monitoring is feasible and useful in patient management. PbtO2 numbers reflect cerebral blood flow and oxygen diffusion. Thus, continuous monitoring of PbtO2 yields important information about both the brain and the lung. The preclinical and clinical studies demonstrating these findings are discussed. In this review, we demonstrate that patient management in a PbtO2-directed fashion is not the sole answer to the problem of treating traumatic brain injury but is an important adjunct to the armamentarium of multimodal neuromonitoring.

Circadian variability of the initial Glasgow Coma Scale score in traumatic brain injury patients

Introduction The Glasgow Coma Scale (GCS) score is the primary method of assessing consciousness after traumatic brain injury (TBI), and the clinical standard for classifying TBI severity. There is scant literature discerning the influence of circadian rhythms or emergency department (ED) arrival hour on this important clinical tool. Methods Retrospective cohort analysis of adult patients suffering blunt TBI using the National Sample Program of the National Trauma Data Bank, years 2003–2006. ED arrival GCS score was characterized by midday (10 a.m.–4 p.m.) and midnight (12 a.m.–6 a.m.) cohorts (N=24548). Proportions and standard errors are reported for descriptive data. Multivariable regressions using odds ratios (OR), mean differences (B), and their associated 95% confidence intervals [CI] were performed to assess associations between ED arrival hour and GCS score. Statistical significance was assessed at p<0.05. Results Patients were 42.48±0.13-years-old and 69.5% male. GCS score was 12.68±0.13 (77.2% mild, 5.2% moderate, 17.6% severe-TBI). Overall, patients were injured primarily via motor vehicle accidents (52.2%) and falls (24.2%), and 85.7% were admitted to hospital (33.5% ICU). Injury severity score did not differ between day and nighttime admissions. Nighttime admissions associated with decreased systemic comorbidities (p<0.001) and increased likelihood of alcohol abuse and drug intoxication (p<0.001). GCS score demonstrated circadian rhythmicity with peak at 12 p.m. (13.03±0.08) and nadir at 4am (12.12±0.12). Midnight patients demonstrated lower GCS (12 a.m.–6 a.m.: 12.23±0.04; 10 a.m.–4 p.m.: 12.95±0.03, p<0.001). Multivariable regression adjusted for demographic and injury factors confirmed that midnight-hours independently associated with decreased GCS (B=−0.29 [−0.40, −0.19]). In patients who did not die in ED or go directly to surgery (N=21862), midnight-hours (multivariable OR 1.73 [1.30–2.31]) associated with increased likelihood of ICU admission; increasing GCS score (per-unit OR 0.82 [0.80–0.83]) associated with decreased odds. Notably, the interaction factor ED GCS score*ED arrival hour independently demonstrated OR 0.96 [0.94–0.98], suggesting that the influence of GCS score on ICU admission odds is less important at night than during the day. Conclusions Nighttime TBI patients present with decreased GCS scores and are admitted to ICU at higher rates, yet have fewer prior comorbidities and similar systemic injuries. The interaction between nighttime hours and decreased GCS score on ICU admissions has important implications for clinical assessment/triage.

182 Ultra-Early (<12 Hours) Decompression Improves Recovery After Spinal Cord Injury Compared to Early (12-24 Hours) Decompression.

INTRODUCTION:Spinal cord injury (SCI) is a devastating condition with very few treatment options. Surgical decompression of the spine after injury has been shown to improve outcomes; however, the optimal timing of surgery is a matter of debate.METHODS:We collected data from 78 patients with SCI. Bas

Do Meningioma Patients Benefit from Antiepileptic Drug Treatment

T w a s t n p C haichana et al. present an interesting article exploring the risk factors associated with preoperative and postoperative seizures in patients with World Health Organization WHO) grade I meningiomas. They show results of a multivariate nalysis of factors from 626 patients who underwent surgical esection for meningioma. They conclude that preoperatively, atients at risk for seizures include those with poor functional status Karnofsky Performance Score 80), absence of signs of increased ntracranial pressure at presentation, and those patients with umors that show significant cerebral edema. From their analysis hey also conclude that patients likely to have continued seizures espite surgery and therapeutic antiepileptic drug (AED) treatent include those patients with uncontrolled preoperative seiures, and patients whose tumors are located parasagittaly or long the sphenoid wing.

Implantation of Neuronal Stem Cells Enhances Object Recognition without Increasing Neurogenesis after Lateral Fluid Percussion Injury in Mice

Cognitive deficits after traumatic brain injury (TBI) are debilitating and contribute to the morbidity and loss of productivity of over 10 million people worldwide. Cell transplantation has been linked to enhanced cognitive function after experimental traumatic brain injury, yet the mechanism of recovery is poorly understood. Since the hippocampus is a critical structure for learning and memory, supports adult neurogenesis, and is particularly vulnerable after TBI, we hypothesized that stem cell transplantation after TBI enhances cognitive recovery by modulation of endogenous hippocampal neurogenesis. We performed lateral fluid percussion injury (LFPI) in adult mice and transplanted embryonic stem cell-derived neural progenitor cells (NPC). Our data confirm an injury-induced cognitive deficit in novel object recognition, a hippocampal-dependent learning task, which is reversed one week after NPC transplantation. While LFPI alone promotes hippocampal neurogenesis, as revealed by doublecortin immunolabeling of immature neurons, subsequent NPC transplantation prevents increased neurogenesis and is not associated with morphological maturation of endogenous injury-induced immature neurons. Thus, NPC transplantation enhances cognitive recovery early after LFPI without a concomitant increase in neuron numbers or maturation.