Early and Prolonged Mild Hypothermia in Patients with Poor-Grade Subarachnoid Hemorrhage: A Pilot Study.

We assessed the feasibility of therapeutic early and prolonged mild hypothermia (MH) in patients with poor-grade subarachnoid hemorrhage (SAH). A retrospective pilot study was conducted for poor-grade SAH patients at two university hospitals from March 2015 to December 2018 who had received MH immediately after coil embolization and maintained a target temperature of 34-35°C for 5 days. A matched controlled design at a 1:2 ratio was used to compare MH therapy outcomes. The primary goal was to assess the two groups' severe functional outcomes at discharge defined as a modified Rankin Scale score of 4-6. The secondary aim was to assess mortality and severe vasospasm depending upon MH. A binary logistic regression analysis was performed to identify relevant risk factors for the outcomes. A total of 54 patients (18 with MH treatment and 36 without MH treatment) were included. Severe functional outcome was significantly decreased in poor-grade SAH patients with MH (n = 7, 38.9%) than those without MH (n = 25, 69.4%; p = 0.031). In patients treated with MH, mortality and severe vasospasm tended to be less common, although the difference was not statistically significant. A binary logistic regression analysis revealed that early and prolonged MH (odds ratio [OR] = 0.156, 95% confidence intervals [CI]: 0.037-0.644) and severe vasospasm (OR = 5.593, 95% CI: 1.372-22.812) were risk factors for severe functional outcomes. This study shows potential therapeutic effect of early and prolonged MH treatment in poor-grade SAH patients. A randomized controlled study with a large number of patients is warranted in the future.

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment.

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner.

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.