Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation.
Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.
The vanilloid receptor type 1 (TRPV1) plays a pivotal role in modulating thermal, chemical, and inflammatory pain. TRPV1s are expressed in some dorsal horn (DH) neurons, but their contribution, if any, to central pain processing still remains unclear. We studied the effects of 2microM capsaicin-induced TRPV1 activation in organotypically cultured substantia gelatinosa neurons from post-natal (8-12) mice. Capsaicin affected sIPSC frequency (272+/-60% of control, n=14, P<0.02), but not amplitude (131+/-12% of control, n=14, P>0.05) in patch clamp recordings, also in the presence of 50microM AP-5 (frequency: 265+/-69% of control; n=8, P<0.05; amplitude: 156+/-28% of control; n=8, P>0.05). The frequency increase was reduced by TTX (181+/-21% of control; n=12, P<0.05). Pre-administration of I-RTX (1microM), a TRPV1 antagonist, prevented the capsaicin effect (frequency: 149+/-28% of control, P>0.05, n=12; amplitude: 97+/-4% of control, P>0.05, n=12). NADA (1microM), an endovanilloid/endocannabinoid agonist of TRPV1, induced a significant increase of sISPC frequency (191+/-40% of control; n=8, P<0.05) without affecting the amplitude (102+/-6% of control; n=8, P>0.05), and the co-application of two naturally occurring N-acyldopamines, PALDA (5microM) and STEARDA (5microM) that facilitate the effect of TRPV1 agonists, also induced a significant increase of sIPSC frequency (278+/-67% of control, n=6, P<0.05). The presence of TRPV1 protein and mRNA in DH neurons was confirmed by histological (immunocytochemistry, in situ PCR) and biochemical (Western blotting, PCR) procedures. These data show that TRPV1 modulates inhibitory neurotransmission in cultured substantia gelatinosa neurons, and suggest that endogenous agonists can activate the spinal receptors in vivo.
Antimicrobial stewardship programs (ASPs) have been suggested to reduce antimicrobial resistance phenomena in veterinary medicine, as antibiotics are commonly used without microbiological confirmation. The aim of the present study is to design a specific working flow for a tailored antimicrobial treatment in the case of canine and feline urinary tract infections (UTIs). Urine samples were collected by cystocentesis from 16 dogs and 12 cats presenting acute signs of UTI. The therapy was decided according to the minimal inhibitory concentration, and it was possible to monitor 14 dogs and 11 cats. Rescue therapy (amoxicillin and clavulanic acid) was included in emergency cases. Escherichia coli, Proteus mirabilis, and Streptococcus canis were isolated in dogs, and Escherichia coli, Staphylococcus pseudintermedius, and Staphylococcus aureus were isolated in cats. No multidrug-resistant strains were detected, but all Staphylococci were methicillin resistant. Only one cat received rescue therapy, and only one dog was recruited. Dogs were treated with tetracycline (1/14), fluoroquinolones (6/14), beta-lactams (6/14), and gentamicin (1/14), while cats received fluoroquinolones (3/11), nitrofurans (1/11), clindamycin (1/11), and beta-lactams (6/11). The success rate was very high. Our findings are interesting because this is the first ASP in Italy, and it may be used as a model to develop ASPs for other pathologies.
B-cell lymphoma 2 protein (BCL-2) is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs) that are known to undergo massive programmed cell death (apoptosis) during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.
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