Markers for neural degeneration and regeneration: novel highly sensitive methods for the measurement of thrombin and activated protein C in human cerebrospinal fluid.

Inflammation and coagulation are tightly interconnected in the pathophysiology of neuronal diseases. Thrombin, a pro-coagulant serine protease is associated with neurodegeneration and its indirect inhibitor, activated protein C (aPC), is considered neuroprotective. While levels of thrombin and aPC activity are readily measured in the blood, similar assays in the cerebrospinal fluid (CSF) have not been described. The aim of this study was to establish a specific and sensitive enzymatic assay to measure both thrombin and aPC activity in the CSF. CSF was collected from 14 patients with suspected normal pressure hydrocephalus served as a control group, while seven patients with central nervous system infections served as an acute neuro-inflammatory study group and one sample of CSF following traumatic lumbar puncture served as a positive control. Thrombin and aPC activities were measured by fluorescence released by specific proteolytic cleavage in the presence of endopeptidase and amino-peptidase inhibitors to ensure specificity. Specificity of the method was verified by thrombin and serine-protease inhibitors N-alpha-((2-naphthylsulfinyl)glycyl)-DL-p-amidinophenylalanylpiperidine and phenylmethanesulfonyl fluoride. Inhibition of thrombin activity by CSF samples and levels of specific thrombin inhibitors were also assessed. Thrombin and aPC activities were reliably measured and were significantly higher in the CSF of patients with central nervous system infections compared to normal pressure hydrocephalus controls, suggesting the involvement of these factors in neuro-inflammation. CSF thrombin activity levels in the presence of known thrombin concentration were high in patients with central nervous system infections, and low in normal pressure hydrocephalus patients. Quantification of endogenous thrombin inhibitors protease nexin 1, amyloid precursor protein and anti-thrombin III in CSF by western blot indicated a significant elevation of amyloid precursor protein in infectious CSF. In conclusion, this study describes a novel and sensitive assay aimed at the detection of thrombin and aPC activity in CSF. This method may be useful for measuring these factors that reflect degenerative and protective influences of coagulation on neurological disorders. The study procedure was approved by the Ethics Committee of the Chaim Sheba Medical Center (approval No. 4245-17-SMC) on October 18, 2018.

Unexpected role of stress as a possible resilience mechanism upon mild traumatic brain injury (mTBI) in mice.

INTRODUCTION: Mild traumatic brain injury (mTBI) is common and associated with cognitive impairment. Stress and mTBI are known to modulate the neural function. The present study aims at exploring the effect of prior stress exposure on cognitive function following mTBI. METHODS: Eight weeks old male ICR mice were subjected to either stress induced by forced swimming stress alone, stress followed by an immediate mTBI, or stress followed by 30 min break and then mTBI. We had two control groups: SHAM group - a control group which was not exposed to stress nor to mTBI and control mTBI group - a control group which was exposed only to TBI with no stress. Mice were weighed prior and at 12, 24 h and 1 week following interventions. Motor evaluation was conducted by rotarod. Behavioral changes were evaluated using open field, Y maze, elevated plus maze and staircase tests, at 12 h and 1 week following interventions. Brain levels of NMDAR subunits (R1, R2A, R2B), GABABR1, glucocorticoid and mineralocorticoid receptors (GR, MR) were evaluated using western blot. RESULTS: Stress alone, mTBI alone, and stress followed by immediate mTBI resulted in a significant weight loss compared to control (p < 0.05). Stress 30 min prior to mTBI had a protective effect on weight (p = 0.14 compared to control). The stress and mTBI alone groups showed reduced time at the center of the open field arena 1 week after intervention (p < 0.05 for both). Time in the novel arm of the Y maze was significantly shorter in the mTBI and stress followed by delayed mTBI (p = 0.02). Immediate stress prior to mTBI had normalized times in the novel arm (p = 0.95 compared to control). Combination of stress and mTBI significantly modified NMDAR subunits levels (increased NMDAR1, p < 0.008, decreased NMDAR2A p = 0.02) as well as increased MR levels (p = 0.04). CONCLUSION: Exposure to stress prior to mTBI may improve the cognitive consequences of mTBI. These data may point towards a novel, unexpected role of stress as a possible resilience mechanism in the setting of mTBI.

A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF.

BACKGROUND: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. METHODS: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. RESULTS: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl2. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. CONCLUSIONS: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF.