ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.

Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1-/- mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.

Intercellular Adhesion Molecule-1-Induced Posttraumatic Brain Injury Neuropathology in the Prefrontal Cortex and Hippocampus Leads to Sensorimotor Function Deficits and Psychological Stress.

Intercellular adhesion molecule-1 (ICAM-1) promotes adhesion and transmigration of circulating leukocytes across the blood-brain barrier (BBB). Traumatic brain injury (TBI) causes transmigrated immunocompetent cells to release mediators [function-associated antigen (LFA)-1 and macrophage-1 antigen (Mac-1)] that stimulate glial and endothelial cells to express ICAM-1 and release cytokines, sustaining neuroinflammation and neurodegeneration. Although a strong correlation exists between TBI-mediated inflammation and impairment in functional outcome following brain trauma, the role of ICAM-1 in impairing functional outcome by inducing neuroinflammation and neurodegeneration after TBI remains inconclusive. The experimental TBI was induced in vivo by fluid percussion injury (FPI; 10 and 20 psi) in wild-type (WT) and ICAM-1-/- mice and in vitro by stretch injury (3 psi) in brain endothelial cells. We manipulate ICAM-1 pharmacologically and genetically and conducted several biochemical analyses to gain insight into the mechanisms underlying ICAM-1-mediated neuroinflammation and performed rotarod, grid-walk, sucrose preference, and light-dark tests to assess functional outcome. TBI-induced ICAM-1-mediated neuroinflammation and cell death occur via LFA-1 or Mac-1 signaling pathways that rely on oxidative stress, matrix metalloproteinase (MMP), and vascular endothelial growth factor (VEGF) pathways. The deletion or blocking of ICAM-1 resulted in a better outcome in attenuating neuroinflammation and cell death as marked by the markers such as NF-kB, IL-1ß, TNF-α, cleaved-caspase-3 (cl-caspase-3), Annexin V, and by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Trypan blue staining. ICAM-1 deletion in TBI improves sensorimotor, depression, and anxiety-like behavior with significant upregulation of norepinephrine (NE), dopamine (DA) D1 receptor (DAD1R), serotonin (5-HT)1AR, and neuropeptide Y (NPY). This study could establish the significance of ICAM-1 as a novel therapeutic target against the pathophysiology to establish functional recovery after TBI.

Enhanced glutamate, IP3 and cAMP activity in the cerebral cortex of unilateral 6-hydroxydopamine induced Parkinson's rats: effect of 5-HT, GABA and bone marrow cell supplementation.

Parkinson's disease is characterized by progressive cell death in the substantia nigra pars compacta, which leads to dopamine depletion in the striatum and indirectly to cortical dysfunction. Increased glutamatergic transmission in the basal ganglia is implicated in the pathophysiology of Parkinson's disease and glutamate receptor mediated excitotoxicity has been suggested to be one of the possible causes of the neuronal degeneration. In the present study, the effects of serotonin, gamma-aminobutyric acid and bone marrow cells infused intranigrally to substantia nigra individually and in combination on unilateral 6-hydroxydopamine induced Parkinson's rat model was analyzed. Scatchard analysis of total glutamate and NMDA receptor binding parameters showed a significant increase in Bmax (P < 0.001) in the cerebral cortex of 6-hydroxydopamine infused rat compared to control. Real Time PCR amplification of NMDA2B, mGluR5, bax, and ubiquitin carboxy-terminal hydrolase were up regulated in cerebral cortex of 6-hydroxydopamine infused rats compared to control. Gene expression studies of GLAST, ά-Synuclien and Cyclic AMP response element-binding protein showed a significant (P < 0.001) down regulation in 6-OHDA infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies. Serotonin and GABA along with bone marrow cells in combination showed reversal of glutamate receptors and behaviour abnormality shown in the Parkinson's rat model. The therapeutic significance in Parkinson's disease is of prominence.

Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and Aegle marmelose.

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT(2A) receptor subtype, gene expression studies on 5-HT(2A), 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B(max) and Kd showed a significant decrease (p < 0.001) whereas 5-HT(2A) receptor binding parameters Bmax showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in Kd in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT(2A), 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B(max), Kd of 5-HT, 5-HT(2A) and gene expression of 5-HT(2A), 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT(2A) and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.