Gut Microbiota as a Therapeutic Target to Ameliorate the Biochemical, Neuroanatomical, and Behavioral Effects of Traumatic Brain Injuries.

Current efficacious treatments for traumatic brain injury (TBI) are lacking. Establishment of a protective gut microbiota population offers a compelling therapeutic avenue, as brain injury induces disruptions in the composition of the gut microbiota, i.e., gut dysbiosis, which has been shown to contribute to TBI-related neuropathology and impaired behavioral outcomes. The gut microbiome is involved in the modulation of a multitude of cellular and molecular processes fundamental to the progression of TBI-induced pathologies including neuroinflammation, blood brain barrier permeability, immune system response, microglial activation, and mitochondrial dysfunction, as well as intestinal motility and permeability. Additionally, gut dysbiosis further aggravates behavioral impairments in animal models of TBI and spinal cord injury, as well as negatively affects health outcomes in murine stroke models. Recent studies indicate that microbiota transplants and probiotics ameliorate neuroanatomical damage and functional impairments in animal models of stroke and spinal cord injury. In addition, probiotics have been shown to reduce the rate of infection and time spent in intensive care of hospitalized patients suffering from brain trauma. Perturbations in the composition of the gut microbiota and its metabolite profile may also serve as potential diagnostic and theragnostic biomarkers for injury severity and progression. This review aims to address the etiological role of the gut microbiome in the biochemical, neuroanatomical, and behavioral/cognitive consequences of TBI, as well as explore the potential of gut microbiome manipulation in the form of probiotics as an effective therapeutic to ameliorate TBI-induced pathology and symptoms.

Age-Related Susceptibility to Epileptogenesis and Neuronal Loss in Male Fischer Rats Exposed to Soman and Treated With Medical Countermeasures.

Elderly individuals compose a large percentage of the world population; however, few studies have addressed the efficacy of current medical countermeasures (MCMs) against the effects of chemical warfare nerve agent exposure in aged populations. We evaluated the efficacy of the anticonvulsant diazepam in an old adult rat model of soman (GD) poisoning and compared the toxic effects to those observed in young adult rats when anticonvulsant treatment is delayed. After determining their respective median lethal dose (LD50) of GD, we exposed young adult and old adult rats to an equitoxic 1.2 LD50 dose of GD followed by treatment with atropine sulfate and the oxime HI-6 at 1 min after exposure, and diazepam at 30 min after seizure onset. Old adult rats that presented with status epilepticus were more susceptible to developing spontaneous recurrent seizures (SRSs). Neuropathological analysis revealed that in rats of both age groups that developed SRS, there was a significant reduction in the density of mature neurons in the piriform cortex, thalamus, and amygdala, with more pronounced neuronal loss in the thalamus of old adult rats compared with young adult rats. Furthermore, old adult rats displayed a reduced density of cells expressing glutamic acid decarboxylase 67, a marker of GABAergic interneurons, in the basolateral amygdala and piriform cortex, and a reduction of astrocyte activation in the piriform cortex. Our observations demonstrate the reduced effectiveness of current MCM in an old adult animal model of GD exposure and strongly suggest the need for countermeasures that are more tailored to the vulnerabilities of an aging population.

Neuroinflammatory and autonomic mechanisms in diabetes and hypertension.

Interdisciplinary studies in the research fields of endocrinology and immunology show that obesity-associated overnutrition leads to neuroinflammatory molecular changes, in particular in the hypothalamus, chronically causing various disorders known as elements of metabolic syndrome. In this process, neural or hypothalamic inflammation impairs the neuroendocrine and autonomic regulation of the brain over blood pressure and glucose homeostasis as well as insulin secretion, and elevated sympathetic activation has been appreciated as a critical mediator. This review describes the involved physiology and mechanisms, with a focus on glucose and blood pressure balance, and suggests that neuroinflammation employs the autonomic nervous system to mediate the development of diabetes and hypertension.

Mapping dopaminergic deficiencies in the substantia nigra/ventral tegmental area in schizophrenia.

Previous work from our laboratory showed deficits in tyrosine hydroxylase protein expression within the substantia nigra/ventral tegmental area (SN/VTA) in schizophrenia. However, little is known about the nature and specific location of these deficits within the SN/VTA. The present study had two aims: (1) test if tyrosine hydroxylase deficits could be explained as the result of neuronal loss; (2) assess if deficits in tyrosine hydroxylase are sub-region specific within the SN/VTA, and thus, could affect specific dopaminergic pathways. To achieve these objectives: (1) we obtained estimates of the number of dopaminergic neurons, total number of neurons, and their ratio in matched SN/VTA schizophrenia and control samples; (2) we performed a qualitative assessment in SN/VTA schizophrenia and control matched samples that were processed simultaneously for tyrosine hydroxylase immunohistochemistry. We did not find any significant differences in the total number of neurons, dopaminergic neurons, or their ratio. Our qualitative study of TH expression showed a conspicuous decrease in labeling of neuronal processes and cell bodies within the SN/VTA, which was sub-region specific. Dorsal diencephalic dopaminergic populations of the SN/VTA presented the most conspicuous decrease in TH labeling. These data support the existence of pathway-specific dopaminergic deficits that would affect the dopamine input to the cortex without significant neuronal loss. Interestingly, these findings support earlier reports of decreases in tyrosine hydroxylase labeling in the target areas for this dopaminergic input in the prefrontal and entorhinal cortex. Finally, our findings support that tyrosine hydroxylase deficits could contribute to the hypodopaminergic state observed in cortical areas in schizophrenia.

Assessment of cytochrome C oxidase dysfunction in the substantia nigra/ventral tegmental area in schizophrenia.

Perturbations in metabolism are a well-documented but complex facet of schizophrenia pathology. Optimal cellular performance requires the proper functioning of the electron transport chain, which is constituted by four enzymes located within the inner membrane of mitochondria. These enzymes create a proton gradient that is used to power the enzyme ATP synthase, producing ATP, which is crucial for the maintenance of cellular functioning. Anomalies in a single enzyme of the electron transport chain are sufficient to cause disruption of cellular metabolism. The last of these complexes is the cytochrome c oxidase (COX) enzyme, which is composed of thirteen different subunits. COX is a major site for oxidative phosphorylation, and anomalies in this enzyme are one of the most frequent causes of mitochondrial pathology. The objective of the present report was to assess if metabolic anomalies linked to COX dysfunction may contribute to substantia nigra/ventral tegmental area (SN/VTA) pathology in schizophrenia. We tested COX activity in postmortem SN/VTA from schizophrenia and non-psychiatric controls. We also tested the protein expression of key subunits for the assembly and activity of the enzyme, and the effect of antipsychotic medication on subunit expression. COX activity was not significantly different between schizophrenia and non-psychiatric controls. However, we found significant decreases in the expression of subunits II and IV-I of COX in schizophrenia. Interestingly, these decreases were observed in samples containing the entire rostro-caudal extent of the SN/VTA, while no significant differences were observed for samples containing only mid-caudal regions of the SN/VTA. Finally, rats chronically treated with antipsychotic drugs did not show significant changes in COX subunit expression. These findings suggest that COX subunit expression may be compromised in specific sub-regions of the SN/VTA (i.e. rostral regions), which may lead to a faulty assembly of the enzyme and a greater vulnerability to metabolic insult.

An accurate method for the quantification of cytochrome C oxidase in tissue sections.

Cytochrome oxidase (COX) is the enzyme that constitutes the last step of the mitochondrial electron transport chain for the production of ATP. Measurement of COX activity can be achieved by histochemistry, thus providing information about the metabolic status of the brain. Brain regions with high metabolism will present high COX activity in histochemistry assays and vice versa. Using histochemistry versus biochemistry to assess COX activity presents the advantage of providing a map of the differences in metabolism in discrete brain regions. Moreover, COX histochemistry allows quantifying the activity of a particular brain region, by converting units of optical density into units of activity. In the present work we have devised a methodology that allows not only quantifying differences in COX activity between groups, but also quantifying the amount of COX present in brain tissue sections, by directly relating optical density (OD) measurements to cytochrome C oxidase concentration, something that traditionally is achieved by the use of western blot. For this purpose we created a set of standards of known concentration of COX that were affixed to a nitrocellulose membrane, and this membrane was incubated together with the tissue sections in which COX activity was assessed. A standard curve was created using a gradient of different concentrations of purified bovine heart cytochrome oxidase (from 2µg to 0.1µg in intervals of 0.25µg). This standard curve allowed us to detect changes in optical density as low as 5%, and relate these OD differences with known concentrations of cytochrome C oxidase.

Dual use of immunohistochemistry for film densitometry and light microscopy.

In the present study, we applied the principles of immunoblotting and light microscopy immunohistochemistry to develop a combined methodology that allows obtaining optical density data in films, as well as morphological and protein distribution data on slides using the same brain tissue section, thus maximizing the data obtained from a single sample. This is especially important when experiments are performed using very valuable or unique tissue samples, which is a very common case in the study of the human brain. The ideal methodology should combine the possibility of measuring levels of expression of a marker, and the capability to map accurately the distribution of that marker in the region of interest. To achieve this, two things are required: first, the technique needs to be sensitive enough to obtain optical density or intensity measurements of the marker, and second, a good preservation of the tissue is needed for the study of distribution patterns and morphological analysis. Here we show that our combined methodology produced reliable results for different tissue preservation conditions (fresh-frozen and fixed tissue), in different species (rat and human), in different brain areas (substantia nigra and striatum), and for the detection of different markers (tyrosine hydroxylase and µ-opioid receptor). This methodology also combines the accuracy of optical density data acquisition in film with obtaining histological slides from the same sample. In summary, the methodology proposed here is very versatile and does not require the use of specialized equipment, other than the routine equipment present in an anatomy laboratory.

Dopamine pathology in schizophrenia: analysis of total and phosphorylated tyrosine hydroxylase in the substantia nigra.

INTRODUCTION: Despite the importance of dopamine neurotransmission in schizophrenia, very few studies have addressed anomalies in the mesencephalic dopaminergic neurons of the substantia nigra/ventral tegmental area (SN/VTA). Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the production of dopamine, and a possible contributor to the anomalies in the dopaminergic neurotransmission observed in schizophrenia. OBJECTIVES: In this study, we had three objectives: (1) Compare TH expression (mRNA and protein) in the SN/VTA of schizophrenia and control postmortem samples. (2) Assess the effect of antipsychotic medications on the expression of TH in the SN/VTA. (3) Examine possible regional differences in TH expression anomalies within the SN/VTA. METHODS: To achieve these objectives three independent studies were conducted: (1) A pilot study to compare TH mRNA and TH protein levels in the SN/VTA of postmortem samples from schizophrenia and controls. (2) A chronic treatment study was performed in rodents to assess the effect of antipsychotic medications in TH protein levels in the SN/VTA. (3) A second postmortem study was performed to assess TH and phosphorylated TH protein levels in two types of samples: schizophrenia and control samples containing the entire rostro-caudal extent of the SN/VTA, and schizophrenia and control samples containing only mid-caudal regions of the SN/VTA. RESULTS AND CONCLUSION: Our studies showed impairment in the dopaminergic system in schizophrenia that could be mainly (or exclusively) located in the rostral region of the SN/VTA. Our studies also showed that TH protein levels were significantly abnormal in schizophrenia, while mRNA expression levels were not affected, indicating that TH pathology in this region may occur posttranscriptionally. Lastly, our antipsychotic animal treatment study showed that TH protein levels were not significantly affected by antipsychotic treatment, indicating that these anomalies are an intrinsic pathology rather than a treatment effect.

Neurochemical characterization of the tree shrew dorsal striatum.

The striatum is a major component of the basal ganglia and is associated with motor and cognitive functions. Striatal pathologies have been linked to several disorders, including Huntington's, Tourette's syndrome, obsessive-compulsive disorders, and schizophrenia. For the study of these striatal pathologies different animal models have been used, including rodents and non-human primates. Rodents lack on morphological complexity (for example, the lack of well defined caudate and putamen nuclei), which makes it difficult to translate data to the human paradigm. Primates, and especially higher primates, are the closest model to humans, but there are ever-increasing restrictions to the use of these animals for research. In our search for a non-primate animal model with a striatum that anatomically (and perhaps functionally) can resemble that of humans, we turned our attention to the tree shrew. Evolutionary genetic studies have provided strong data supporting that the tree shrews (Scadentia) are one of the closest groups to primates, although their brain anatomy has only been studied in detail for specific brain areas. Morphologically, the tree shrew striatum resembles the primate striatum with the presence of an internal capsule separating the caudate and putamen, but little is known about its neurochemical composition. Here we analyzed the expression of calcium-binding proteins, the presence and distribution of the striosome and matrix compartments (by the use of calbindin, tyrosine hydroxylase, and acetylcholinesterase immunohistochemistry), and the GABAergic system by immunohistochemistry against glutamic acid decarboxylase and Golgi impregnation. In summary, our results show that when compared to primates, the tree shrew dorsal striatum presents striking similarities in the distribution of most of the markers studied, while presenting some marked divergences when compared to the rodent striatum.