Maternal and Cord Anti-SARS-CoV-2-Spike IgG following COVID-19 Vaccination versus Infection during Pregnancy: A Prospective Study, Israel October 2021-March 2022.

OBJECTIVE: Defining how pregnant women respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination is critical to optimize vaccination strategies that protect mother and infant at the epidemic. This study aimed to compare anti-SARS-CoV-2-spike immunoglobulin G (IgG) of vaccinated versus infected women and to determine the optimal timing of maternal vaccination during pregnancy at the time of epidemic. STUDY DESIGN: We collected maternal/cord blood at delivery (October 2021-March 2022) and measured anti-SARS-CoV-2-spike IgG geometric mean concentrations (IgG-GMCs) using a quantitative immunoassay. We compared groups according to timing and number of doses and correlated maternal and fetal IgG levels. We described the proportion of women with IgG levels above the 150 AU/mL positivity threshold according to the timing of infection/vaccination and performed a subanalysis for maternal IgG-GMC levels pre- and during the Omicron wave. RESULTS: We included 238 vaccinated women, 125 who received two doses and 113 three doses, and 48 unvaccinated infected women. All groups infected/vaccinated in the second or third trimester had an IgG-GMC above the positivity threshold. Third-trimester vaccination (second/third dose) resulted in higher maternal and cord-blood IgG-GMC compared to the second trimester (maternal-IgG: 102,32 vs. 4,325 AU/mL, p < 0.001; cord-IgG: 12,113 vs. 8,112 AU/mL, p < 0.001). Compared with infected-only women, a higher proportion of vaccinated women with ≥2 doses and their newborns had IgG levels above the positivity threshold at all time points. In vaccinated women, there were higher maternal IgG-GMC levels during the Omicron wave than pre-Omicron. CONCLUSION: At the time of epidemic, receiving an additional COVID-19 vaccine dose in the third trimester resulted in a higher IgG-GMC compared to the second trimester. Relatively higher levels of maternal and cord IgG-GMC were achieved following vaccination than infection. Women infected during or before the first trimester might benefit from an additional third-trimester dose to prevent peripartum infection and to passively immunize their newborn. The higher levels of maternal IgG-GMC in the Omicron period are suggestive of hybrid immunity. KEY POINTS: · Higher maternal anti-SARS-IgGs in vaccinated → infected.. · Higher cord anti-SARS-IgGs in vaccinated → infected.. · Third-trimester vaccine resulted in high-cord IgG levels..

Baseline moderate-range albuminuria is associated with protection against severe COVID-19 pneumonia.

BACKGROUND: Diabetes mellitus is considered a leading contributor to severe coronavirus disease 2019 (COVID-19). AIM: To characterize differences between hospitalized diabetic patients with vs without COVID-19, and parameters associated with COVID-19 severity for prediction. METHODS: This case-control study included 209 patients with type 2 diabetic mellitus hospitalized at the Galilee Medical Center (Nahariya, Israel) and recruited between September 2020 and May 2021, 65 patients with COVID-19 infection in dedicated wards and 144 COVID-19-negative patients in internal medicine wards hospitalized due to other reasons. Clinical parameters - including age, type of antiglycemic medications, presence of retinopathy, smoking history, body mass index (BMI), glycosylated hemoglobin, maximum neutrophil:lymphocyte ratio (NLRmax), C-reactive protein (CRP), estimated glomerular filtration rate (eGFR), and albumin (blood and urine) - were compared between the two primary patient groups, and then between COVID-19-negative patients hospitalized due to infectious vs non-infectious disease. Finally, we explored which parameters were associated with severe COVID-19 pneumonia. RESULTS: COVID-19-negative patients were older (63.9 ± 9.9 vs 59.8 ± 9.2, P = 0.005), and had longer duration of diabetes (P = 0.031), lower eGFR (P = 0.033), higher albumin (P = 0.026), lower CRP (P < 0.001), greater smoking prevalence (P < 0.001), and more baseline albuminuria (54.9% vs 30.8%, P = 0.005) at admission; 70% of COVID-19 patients with albuminuria had moderate-range albuminuria (albumin:creatinine 30-300 mg/g). Most of the patients with albuminuria had chronic kidney disease stage II (CKD II). Oral antiglycemic therapies were not significantly different between the two groups. Multivariable logistic regression showed that higher BMI was significantly associated with severe COVID-19 (OR 1.24, 95%CI: 1.01-1.53, P = 0.04), as was higher NLRmax (OR 1.2, 95%CI: 1.06-1.37, P = 0.005). Surprisingly, pre-hospitalization albuminuria, mostly moderate-range, was associated with reduced risk (OR 0.09, 95%CI: 0.01-0.62, P = 0.015). Moderate-range albuminuria was not associated with bacterial infections. CONCLUSION: Moderate-range albuminuria in COVID-19-positive diabetic patients with CKD II is associated with less severe COVID-19. Further studies should explore this potential biomarker for risk of COVID-19-related deterioration and early interventions.

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult's leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology.

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology.

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

Serum albumin levels and inflammation.

Albumin is the most abundant protein in the plasma and has a regulatory role in the distribution of body fluids, acid-base physiology, and binding of essential components in the bloodstream. C-reactive protein (CRP) is produced by hepatocytes and is commonly used to assess inflammation. It was previously noted that acute-phase concentrations of proteins, such as CRP, tend to rise in inflammatory conditions, while albumin concentrations tend to decline. This study assessed the correlation between albumin levels and various inflammatory indices (CRP, WBC, PLT) of patients hospitalized at the Galilee Medical Center over a period of 3 months. The study population consisted of 4434 patients, ages 18-107 years (mean: 52 years), of whom 60% were female. A negative correlation between albumin and CRP levels (r = -0.311) was identified, as well as between albumin and white blood cells levels (r = -0.157). Positive correlations were found between albumin and platelets levels (r = 0.084), as well as between albumin and hemoglobin levels (r = 0.513). When considering the three largest departments, the strongest negative correlation between albumin and CRP was identified in the Internal Medicine departments. A linear regression analysis discovered a fairly minor effect of CRP on albumin levels, which only became apparent when CRP levels were extremely high (500 mg/L). The mechanisms underlying this negative correlation still need to be explored.

Subclinical hypothyroidism or isolated high TSH in hospitalized patients with chronic heart-failure and chronic renal-failure.

Sub-clinical hypothyroidism (SCH) is common in heart failure (HF) and advanced renal failure (RF), but it is unclear whether there is a thyroid disease or a transient increase in TSH level. This is a retrospective study of hospitalized patients in medical departments. All patients with SCH and a TSH level up to less than 12 mIU/L were identified. Those who had at least one recurring admission within at least 6 months were included. A change in thyroid function during the last re-admission was determined and classified as an improvement, no change, or worsening of thyroid function. Overall, 126 cases of SCH met the inclusion criteria for re-admission. Analysis of the most recent hospitalization showed that in 100 (79.4%) patients thyroid function improved, in 15 (11.9%) patients thyroid function remained unchanged and only in 11 (8.7%) patients did thyroid function worsen. In most cases, worsening of hypothyroidism was determined by initiation of a low dose levothyroxine treatment. Of the 126 participants, 43 (34.1%) and 22 (17.5%) had a diagnosis of HF and RF (CKD stages 4 and 5), respectively. There was no association between HF or advanced RF and worsening of SCH. No association was found between worsening of hypothyroidism and gender, age, TSH, or creatinine levels in the first hospitalization. A borderline association between elevated CRP levels at first hospitalization and hypothyroidism worsening was found (p = 0.066). Mildly elevated TSH in hospitalized patients with HF and advanced RF is transient and most probably not related to thyroid disease and not associated with age or gender.

The significance of high thyroxine in hospitalized patients with low thyroid-stimulating hormone.

PURPOSE: There is scarce data about the interpretation of high thyroid hormone levels in hospitalized patients. We wished to investigate the significance of high thyroxine (T4) in hospitalized patients with low TSH. METHODS: We conducted a retrospective study of data from patients in nonsurgical departments. Three groups of random patients with low TSH were defined and compared: 123 patients with only high FT4 levels (T4 group), 82 with high FT3 levels with or without high FT4 level (T3 group), and 119 with low FT3 and FT4 level in the lower half of the norm and below (NTIS group). RESULTS: The primary cause of admission in the T4 and NTIS groups was infectious disease, 20.3% and 40.3%, respectively; while in the T3 group it was cardiovascular disease (31.7%). The T4 group but not T3 group had epidemiological and clinical characteristics similar to the NTIS group. The T4 group had a significant correlation between increased CRP levels and decreased FT3 (r = 0.366, p < 0.001) similar to the NTIS group. The T3 group had a borderline correlation between increased FT3 and FT4 levels (r = 0.208, p = 0.061) but the T4 group did not. CONCLUSIONS: The combination of low TSH and high FT4 levels in hospitalized patient is usually caused by nonthyroidal illness combined with drug effects. This thyroid function disturbance is common in hospitalized patients and if the FT3 level is below the middle of the norm, treatment is probably unnecessary.

Cell-free IgG-aggregates in plasma of patients with chronic lymphocytic leukemia cause chronic activation of the classical complement pathway.

Therapy regimens for Chronic lymphocytic leukemia (CLL) commonly include chemotherapy and immunotherapy, which act through complement-mediated-cytotoxicity (CDC) and other mechanisms. CDC depends on several factors, including the availability and activity of the complement classical pathway (CP). Recently, a significant decrease in CP activity was shown to be associated with an immunoglobulin-C5a complex (Ig-C5a) and other markers of chronic CP activation in 40% of the patients. The study focused on the involvement of IgG-hexamers, an established CP activator, in the mechanism of chronic CP activation in CLL. Sera from 51 naïve CLL patients and 20 normal controls were collected. CP and alternative pathway (AP) activities were followed by the complement activity marker sC5b-9. Serum high molecular weight (HMW) proteins were collected by gel-filtration chromatography and their complement activation capacity was assessed. The levels of IgM, another established CP activator, were measured. Data were associated with the presence of Ig-C5a. Baseline levels of activation markers negatively correlated with CP and the AP activities, supporting chronic complement activation. In patients with Ig-C5a, HMW proteins that are not IgM, activated the complement. HMW proteins were identified as IgG-aggregates by affinity binding assays and Western blot analysis. The data indicate chronic CP activation, mediated by cell-free IgG-hexamers as a cause of decreased CP activity in part of the CLL population. This mechanism may affect immunotherapy outcomes due to compromised CP activity and CDC.

Low brain-derived neurotrophic factor protein levels and single-nucleotide polymorphism Val66Met are associated with peripheral neuropathy in type II diabetic patients.

BACKGROUND: Studies by our group demonstrated brain-derived neurotrophic factor (BDNF) levels in blood and BDNF-Val66met-SNP as potential biomarkers in chemotherapy-induced peripheral neuropathy. Here, we evaluate symptoms of peripheral neuropathy (PN) and depression in patients with type II diabetes mellitus in search of an association with serum BDNF levels and the Val66Met-SNP. METHODS: In total, 90 patients enrolled in the study; 23 (25.6%) had known PN, as determined by nerve conduction studies (NCS-PN), and 67 (74.4%) were not diagnosed with PN (U-PN). PN symptoms were assessed and graded in these groups using the total neuropathy score (TNSr) and DN4 scales. Small nerve fiber testing of sensitivity thresholds to cold, warm and hot pain signals was performed using the Q-sense device. Depression was assessed using the PHQ9 questionnaire. BDNF protein levels and Val66Met-SNP were determined with ELISA and Sanger sequencing, respectively. RESULTS: NCS-PN patients showed lower serum BDNF levels alongside significantly higher TNSr, DN4 and PHQ9 scores and lower hot pain sensitivity thresholds as compared to U-PN patients. Patients with Met-BDNF-SNP showed increased TNSr scores and lower hot pain sensitivity thresholds as compared to patients with Val-BDNF-SNP. Depression showed a weaker correlation with sensitivity thresholds to hot pain signals as compared to TNSr and DN4 scores. CONCLUSIONS: Diminished peripheral BDNF resources and Met-BDNF-SNP genotype are associated with augmented symptoms of PN in patients with type II diabetes mellitus. Sensitivity thresholds to hot pain signals may be less influenced by depression and possibly more accurately detect PN symptoms in diabetic patients.

The Role of Alpha 2 Macroglobulin in IgG-Aggregation and Chronic Activation of the Complement System in Patients With Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.

Reduction of Traumatic Brain Damage by Tspo Ligand Etifoxine.

Experimental studies have shown that ligands of the 18 kDa translocator protein can reduce neuronal damage induced by traumatic brain injury by protecting mitochondria and preventing metabolic crisis. Etifoxine, an anxiolytic drug and 18 kDa translocator protein ligand, has shown beneficial effects in the models of peripheral nerve neuropathy. The present study investigates the potential effect of etifoxine as a neuroprotective agent in traumatic brain injury (TBI). For this purpose, the effect of etifoxine on lesion volume and modified neurological severity score at 4 weeks was tested in Sprague-Dawley adult male rats submitted to cortical impact contusion. Effects of etifoxine treatment on neuronal survival and apoptosis were also assessed by immune stains in the perilesional area. Etifoxine induced a significant reduction in the lesion volume compared to nontreated animals in a dose-dependent fashion with a similar effect on neurological outcome at four weeks that correlated with enhanced neuron survival and reduced apoptotic activity. These results are consistent with the neuroprotective effect of etifoxine in TBI that may justify further translational research.

A C5a-Immunoglobulin complex in chronic lymphocytic leukemia patients is associated with decreased complement activity.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

Associations of Peritoneal Glucose Load With Male Sexual Dysfunction and Depression in Peritoneal Dialysis Patients.

This cross-sectional study examined possible associations of peritoneal glucose load with male sexual dysfunction and depression in peritoneal dialysis patients. Compared to patients with peritoneal glucose load ≤3 g/kg per day, those with load >3 g/kg per day had higher Beck Depression Inventory scores, (18.9 ± 5.4 vs. 11.4 ± 5.8, P = 0.002) and lower International Index of Erectile Function scores, serum total testosterone and DHEA [(15.4 ± 6.4 vs. 45.1 ± 20.7, P < 0.001), (8.5 ± 3.0 vs. 13.9 ± 3.2, P < 0.001), (113.9 ± 58.8 vs. 280.2 ± 128.3, P < 0.001); respectively)]. Of participants with peritoneal glucose load >3 g/kg per day, 84.6% had mild to moderate erectile dysfunction and 92.3% had abnormal Beck Depression Inventory scores. Peritoneal glucose load inversely correlated with International Index of Erectile Function scores (P < 0.001), total serum testosterone (P = 0.002) and serum DHEA (P = 0.001); and directly with Beck Depression Inventory scores (P < 0.001) and serum estradiol (P < 0.001). This study demonstrated higher prevalence of sexual dysfunction, depression and sex hormone disturbances in male peritoneal dialysis patients receiving higher peritoneal glucose load.

The relationship between hydration status, male sexual dysfunction and depression in hemodialysis patients.

BACKGROUND: Disturbances in sexual function are common among dialysis patients. Normal erections require a complex balance of physiological, psychological, emotional, hormonal, neurological and vascular factors. This study examined a possible association of overhydration (OH) with male sexual dysfunction and depression in hemodialysis (HD) patients. PATIENTS AND METHODS: This cross-sectional study assessed hydration status by whole-body bioimpedance spectroscopy in patients on maintenance HD for more than 12 months. Patients were categorized according to OH to extracellular water (ECW) ratio: OH/ECW ratio >0.15 and OH/ECW ratio ≤0.15. Sexual function was assessed using the International Index of Erectile Function (IIEF) score. Psychological status was evaluated using the Beck Depression Inventory (BDI) score. Serum sex hormones were determined. RESULTS: Of 39 stable participants on HD, 53.8% were overhydrated (OH/ECW ratio >0.15) and 46.2% not overhydrated (OH/ECW ratio ≤0.15). Of participants with OH/ECW ratio >0.15, 85.7% had mild to severe ED, and 71.4% had abnormal BDI scores, ranging from mild mood disturbance to severe depression. Compared to patients with OH/ECW ratio ≤0.15, BDI scores, serum estradiol and plasma hsCRP were higher (18.48±8.34 vs 10.61±5.46, p<0.001; 140.10±44.51 vs 126.10±32.26, p=0.034; and, 17.70±12.14 vs 9.76±8.79, p=0.013; respectively) in those with OH/ECW ratio >0.15, while their IIEF score, serum total testosterone and dehydroepiandrosterone (DHEA) were lower (12.81±7.31 vs 41.44±23.79, p<0.001; 8.97±5.43 vs 14.10±8.30, p=0.013; and 85.31±55.14 vs 133.3±95.48, p=0.029; respectively). The OH/ECW ratio correlated inversely with the IIEF score (r=-0.69, p<0.001) and positively with BDI scores (r=0.64, p<0.001). IIEF scores were inversely correlated with BDI scores (r=-0.54, p<0.001). CONCLUSION: OH in HD patients was found to be associated with a higher prevalence of sexual dysfunction and depression, lower serum levels of total testosterone and DHEA, and higher levels of serum estradiol.

[INVOLVEMENT OF EPIGENETIC REGULATION IN CEREBRAL CELL DEATH FOLLOWING TRAUMATIC BRAIN INJURY IN A RAT MODEL].

INTRODUCTION: Traumatic brain injuries (TBI) are a major cause of mortality and disability among young adults. TBI are characterized by primary injury, the result of a mechanical impact to the cranium and a secondary injury, a series of molecular mechanism processes developing thereafter. Cerebral cells modify their gene and protein expression as a result of the injury. Epigenetic modifications have a key role as regulators of gene transcription and may simultaneously be involved in the regulation of the molecular pathways following TBI. However, the mechanisms are unknown. OBJECTIVES: To clarify whether modification in the expression of Histone Acetyl Transferase1 (HAT1) and Histone deacetylase2 (HDAC 2) occurs during secondary brain damage. METHODS: Rat diffused head injury model was used; 72 hours post injury animals were sacrificed and the brains were removed for immunohistochemistry staining with Caspase 3, HAT1 and HDAC2 antibodies. We compared these stains in the perilesional versus the contralateral cortex. RESULTS: An increase of Caspase 3 stained cells were observed in the perilesional cortex. HAT1 expression was elevated and HDAC2 expression reduced in the injured cortex. CONCLUSIONS: TBI induced modifications in the expression of epigenetic factors were concomitant with increases in apoptotic cell death. The mitochondria involved in the apoptotic processes is a target for epigenetic regulation and also influences it at the same time. DISCUSSION: This study contributes to the understanding of epigenetic modification following TBI. Further study on the relationship between mitochondrial activity and epigenetic regulation has to be performed in order to develop novel drugs and therapies for TBI.

Post-traumatic cytotoxic edema is directly related to mitochondrial function.

Cerebral edema represents a major threat following traumatic brain injury. However, therapeutic measures for control of intracranial pressure alone have failed to restore cerebral metabolism and improve neurological outcome. Since mitochondrial damage results in ATP depletion and deactivation of membrane ionic pumps, we hypothesized that modulation of ATP bioavailability may directly affect cytotoxic edema. Intracranial pressure measurements were performed in Sprague-Dawley rats treated by intraperitoneal injection of dimethylsulfoxide (vehicle), cyclosporine A (CsA), or Oligomycin B (OligB) following cortical contusion and further correlated with water content, mitochondrial damage, and electron microscopic assessment of neuronal and axonal edema. As hypothesized, ultra-structural figures of edema closely correlated with intracranial pressure elevation, increased water content and mitochondrial membrane permeabilization expressed by loss of transmembrane mitochondrial potential. Further, mitochondrial damage evidenced ultra-structurally by figures of swollen mitochondria with severely distorted cristae correlated with both cytotoxic edema and mitochondrial dysfunction. Importantly, cerebral edema and mitochondrial impairment were significantly worsened by treatment with OligB, whereas a noticeable improvement could be observed in animals that received injections of CsA. Since OligB and CsA are responsible for symmetrical and opposite effects on oxidative metabolism, these findings support the hypothesis of a causative relationship between edema and mitochondrial function.

Investigation of the mechanisms of neuroprotection mediated by Ro5-4864 in brain injury.

Increasing evidence has established the involvement of the 18-kDa translocator protein (TSPO) in the process of mitochondrial membrane permeabilization and subsequent apoptosis through modulation of the mitochondrial permeability transition pore. Recent studies have shown that treatment with Ro5-4864, a TSPO ligand, resulted in a neuroprotective effect in traumatic brain injury. Yet, the nature of this effect remained uncertain as mature neurons are considered to be lacking the TSPO protein. In order to investigate the mechanism of Ro5-4864-mediated neuroprotection, the neuro-inflammatory and neurosteroid response to cortical injury was tested in sham-operated, vehicle, cyclosporine A (CsA) and Ro5-4864-treated rats. As anticipated, the levels of interleukin 1ß and tumor necrosis factor α, as well as the astrocyte and microglia cellular density in the injured area were all decreased by CsA in comparison with the vehicle group. By contrast, no visible effect could be observed in Ro5-4864-treated animals. None of the groups showed any significant difference with any other in respect with the expression of brain-derived neurotrophic factor. Double immunofluorescence staining with NeuN and TSPO confirmed the absence of TSPO in native neurons though showed clear evidence of co-localization of TSPO in the cytoplasm of NeuN-stained injured neurons. Altogether, this study shows that the neuronal protection mediated by Ro5-4864 in brain injury cannot be solely attributed to an indirect effect of the ligand on glial TSPO but may also represent the consequence of the modulation of upregulated TSPO in injured neurons. This observation may be of importance for future pharmacological research in neurotrauma.