Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment.
Peripheral axotomy in neonatal rats induces neuronal death. We studied the anti-apoptotic protein Bcl-2 and cell death promoter Bax in spinal cord of neonatal rats after sciatic transection and treatment with melatonin, a neuroprotective substance. Pups were unilaterally axotomized at P2 and received melatonin (1 mg/kg; sc) or vehicle 1 h prior to lesion, immediately after, at 1 h, 2 h and then once daily. Rats were sacrificed at 3 h, 6 h, 24 h, 72 h and 5 days postaxotomy. Intact animals were used as controls. Lumbar enlargement was processed for Nissl staining, immunohistochemistry and RT-PCR for Bax or Bcl-2 and TUNEL reaction. Motoneurons (MN) of lesioned (L) and normal (N) sides were counted, and MN survival ratio (MSR=L/N) was calculated. Bax and Bcl-2 showed cytoplasmic immunoreactivity (IR). Bax IR was noticeable in small cells but less evident in MN. In unlesioned pups, some Bax-positive small cells (B+) and TUNEL-positive nuclei (T+) were mainly seen in the dorsal horn. In lesioned animals given vehicle, Bax mRNA levels and numbers of B+ and T+ were increased in comparison with intact controls at 24 h postaxotomy. The basal IR for Bax in MN was not changed by axotomy. Bcl-2 IR was noted in all cells and, like Bcl-2 mRNA, was unaltered after lesion. Melatonin reduced MN loss at 24 h, 72 h and 5 days and T+ at 24 h after lesion but did not interfere with Bax or Bcl-2 expression. These results suggest that (1) sciatic transection at P2 increases Bax mRNA and the amount of B+ and T+ in the lumbar enlargement, (2) Bax IR in immature MN is not altered by axotomy and (3) melatonin protects MN and dorsal horn cells through a mechanism independent of Bax and Bcl-2.
Rogério
Fábio
F
Department of Physiology and Biophysics, State University of Campinas, UNICAMP, 13083-970, Campinas, SP, Brazil.
Jordão
Hamilton
H
Vieira
André Schwambach
AS
Maria
Carla Cristina Judice
CC
Santos de Rezende
Alexandre César
AC
Pereira
Gonçalo Amarante Guimarães
GA
Langone
Francesco
F
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000627:therapeutic use*; D019771:Axotomy / Q000379:methods; D002452:Cell Count / Q000379:methods; D004195:Disease Models, Animal; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D020287:In Situ Nick-End Labeling / Q000379:methods; D008161:Lumbosacral Region; D008550:Melatonin / Q000627:therapeutic use*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D020426:Sciatic Neuropathy / Q000188:drug therapy / Q000473:pathology / Q000503:physiopathology; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
D000975:Antioxidants; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D008550:Melatonin