Chronic Anxiety- and Depression-Like Behaviors Are Associated With Glial-Driven Pathology Following Repeated Blast Induced Neurotrauma.

Long-term neuropsychiatric impairments have become a growing concern following blast-related traumatic brain injury (bTBI) in active military personnel and Veterans. Neuropsychiatric impairments such as anxiety and depression are common comorbidities that Veterans report months, even years following injury. To understand these chronic behavioral outcomes following blast injury, there is a need to study the link between anxiety, depression, and neuropathology. The hippocampus and motor cortex (MC) have been regions of interest when studying cognitive deficits following blast exposure, but clinical studies of mood disorders such as major depressive disorder (MDD) report that these two regions also play a role in the manifestation of anxiety and depression. With anxiety and depression being common long-term outcomes following bTBI, it is imperative to study how chronic pathological changes within the hippocampus and/or MC due to blast contribute to the development of these psychiatric impairments. In this study, we exposed male rats to a repeated blast overpressure (~17 psi) and evaluated the chronic behavioral and pathological effects on the hippocampus and MC. Results demonstrated that the repeated blast exposure led to depression-like behaviors 36 weeks following injury, and anxiety-like behaviors 2-, and 52-weeks following injury. These behaviors were also correlated with astrocyte pathology (glial-fibrillary acid protein, GFAP) and dendritic alterations (Microtubule-Associated Proteins, MAP2) within the hippocampus and MC regions at 52 weeks. Overall, these findings support the premise that chronic glial pathological changes within the brain contribute to neuropsychiatric impairments following blast exposure.

Glial Activation in the Thalamus Contributes to Vestibulomotor Deficits Following Blast-Induced Neurotrauma.

Vestibular impairment has become a frequent consequence following blast-related traumatic brain injury (bTBI) in military personnel and Veterans. Behavioral outcomes such as depression, fear and anxiety are also common comorbidities of bTBI. To accelerate pre-clinical research and therapy developments, there is a need to study the link between behavioral patterns and neuropathology. The transmission of neurosensory information often involves a pathway from the cerebral cortex to the thalamus, and the thalamus serves crucial integrative functions within vestibular processing. Pathways from the thalamus also connect with the amygdala, suggesting thalamic and amygdalar contributions to anxiolytic behavior. Here we used behavioral assays and immunohistochemistry to determine the sub-acute and early chronic effects of repeated blast exposure on the thalamic and amygdala nuclei. Behavioral results indicated vestibulomotor deficits at 1 and 3 weeks following repeated blast events. Anxiety-like behavior assessments depicted trending increases in the blast group. Astrogliosis and microglia activation were observed upon post-mortem pathological examination in the thalamic region, along with a limited glia response in the amygdala at 4 weeks. These findings are consistent with a diffuse glia response associated with bTBI and support the premise that dysfunction within the thalamic nuclei following repeated blast exposures contribute to vestibulomotor impairment.

Heat shock protein 70 is necessary to improve mitochondrial bioenergetics and reverse diabetic sensory neuropathy following KU-32 therapy.

Impaired neuronal mitochondrial bioenergetics contributes to the pathophysiologic progression of diabetic peripheral neuropathy (DPN) and may be a focal point for disease management. We have demonstrated that modulating heat shock protein (Hsp) 90 and Hsp70 with the small-molecule drug KU-32 ameliorates psychosensory, electrophysiologic, morphologic, and bioenergetic deficits of DPN in animal models of type 1 diabetes. The current study used mouse models of type 1 and type 2 diabetes to determine the relationship of changes in sensory neuron mitochondrial bioenergetics to the onset of and recovery from DPN. The onset of DPN showed a tight temporal correlation with a decrease in mitochondrial bioenergetics in a genetic model of type 2 diabetes. In contrast, sensory hypoalgesia developed 10 weeks before the occurrence of significant declines in sensory neuron mitochondrial bioenergetics in the type 1 model. KU-32 therapy improved mitochondrial bioenergetics in both the type 1 and type 2 models, and this tightly correlated with a decrease in DPN. Mechanistically, improved mitochondrial function following KU-32 therapy required Hsp70, since the drug was ineffective in diabetic Hsp70 knockout mice. Our data indicate that changes in mitochondrial bioenergetics may rapidly contribute to nerve dysfunction in type 2 diabetes, but not type 1 diabetes, and that modulating Hsp70 offers an effective approach toward correcting sensory neuron bioenergetic deficits and DPN in both type 1 and type 2 diabetes.

Modulating molecular chaperones improves sensory fiber recovery and mitochondrial function in diabetic peripheral neuropathy.

Quantification of intra-epidermal nerve fibers (iENFs) is an important approach to stage diabetic peripheral neuropathy (DPN) and is a promising clinical endpoint for identifying beneficial therapeutics. Mechanistically, diabetes decreases neuronal mitochondrial function and enhancing mitochondrial respiratory capacity may aid neuronal recovery from glucotoxic insults. We have proposed that modulating the activity and expression of heat shock proteins (Hsp) may be of benefit in treating DPN. KU-32 is a C-terminal Hsp90 inhibitor that improved thermal hypoalgesia in diabetic C57Bl/6 mice but it was not determined if this was associated with an increase in iENF density and mitochondrial function. After 16 weeks of diabetes, Swiss Webster mice showed decreased electrophysiological and psychosensory responses and a >30% loss of iENFs. Treatment of the mice with ten weekly doses of 20mg/kg KU-32 significantly reversed pre-existing deficits in nerve conduction velocity and responses to mechanical and thermal stimuli. KU-32 therapy significantly reversed the pre-existing loss of iENFs despite the identification of a sub-group of drug-treated diabetic mice that showed improved thermal sensitivity but no increase in iENF density. To determine if the improved clinical indices correlated with enhanced mitochondrial activity, sensory neurons were isolated and mitochondrial bioenergetics assessed ex vivo using extracellular flux technology. Diabetes decreased maximal respiratory capacity in sensory neurons and this deficit was improved following KU-32 treatment. In conclusion, KU-32 improved physiological and morphologic markers of degenerative neuropathy and drug efficacy may be related to enhanced mitochondrial bioenergetics in sensory neurons.

Heat shock response and insulin-associated neurodegeneration.

Dysfunctional insulin and insulin-like growth factor-I (IGF-I) signaling contributes to the pathological progression of diabetes, diabetic peripheral neuropathy (DPN), Alzheimer's (AD), Parkinson's (PD) and Huntington's diseases (HD). Despite their prevalence, there are limited therapeutic options available for the treatment of these neurodegenerative disorders. Therefore, establishing a link between insulin/IGF-I and the pathoetiology of these diseases may provide alternative approaches toward their management. Many of the heat shock proteins (Hsps) are well-known molecular chaperones that solubilize and clear damaged proteins and protein aggregates. Recent studies suggest that modulating Hsps may represent a promising therapeutic avenue for improving insulin and IGF-I signaling. Pharmacological induction of the heat shock response (HSR) may intersect with insulin/IGF-I signaling to improve aspects of neurodegenerative phenotypes. Herein, we review the intersection between Hsps and the insulin/IGF systems under normal and pathological conditions. The discussion will emphasize the potential of non-toxic HSR inducers as viable therapeutic agents.

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice.

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

Key residues controlling phenacetin metabolism by human cytochrome P450 2A enzymes.

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

Prehospital 12-lead ECG diagnostic programs.

Early reperfusion significantly reduces mortality and morbidity in patients with acute myocardial infarction [2-6]. Prehospital 12-lead ECG programs significantly decrease time to definitive reperfusion therapy [8-13]. The feasibility and safety of prehospital 12-lead ECG programs are well [figure: see text] established [8,11,13,14]. Additional potential benefits include increased diagnostic accuracy in the prehospital setting [14], providing a comparison ECG to the one obtained in-hospital [15], differentiating arrhythmias [16-18], and sensitive and specific computerized ECG interpretation [31,32]. Prehospital 12-lead ECG diagnostic programs also provide the necessary clinical information to implement system changes or interventions such as prehospital thrombolytic therapy, direct CCU admission, or triage to tertiary cardiac care centers [22,30,34,35]. The information acquired should be used optimally to effect significant improvements in patient care through a well planned and coordinated program.