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Members of the transforming growth factor-␤ (TGF-␤) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-␤ signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-␤ superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-␤1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-␤1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-␤/activinrestricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-␤ superfamily Smad signaling pathways plays a morphogenic role during endochondral bone

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The aims of this study were to clarify the mechanism of cell death by apoptosis in the spinal cord after traumatic injury, and to examine the role of the mitogen-activated protein kinase (MAPK) pathways in the transmission of apoptosis signals. The rat spinal cord, experimentally injured by extradural static weight-compression, was studied by hematoxylin and eosin staining, Nissl-staining, terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) staining, and immunostaining using polyclonal antibodies against Apoptosis Signal-regulating Kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38 MAPK. TUNEL-positive cells were present at all stages studied until 7 days after injury, and percentage positivity for these cells was maximal at 3 days after injury. Electron microscopic analysis revealed the occurrence of apoptosis in both neuronal cells and glial cells. TUNEL-positive glial cells were stained by oligodendrocyte-specific maker. Expression of ASK1 was maximal at 24 h after injury in the gray matter and at 3 days after injury in the white matter. Following the expression of ASK1, activated forms of JNK and p38 were observed in apoptotic cells detected by the TUNEL method. Colocalization of ASK1 and activated JNK or activated p38 was observed in the same cell. These findings suggest the involvement of the stress-activated MAPK pathways including ASK1 in the transmission of apoptosis signals after spinal cord injury.

Object. The aims of this study were to clarify the histological and histochemical changes associated with cell death in the spinal cord after acute traumatic injury and to examine the role of excitatory amino acid release mediated by N-methyl-d- aspartate (NMDA) receptors. Methods. Following laminectomy, the spinal cord in 70 rats was injured at the T-9 level by applying extradural static weight—compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury (SCI) with paralysis of the lower extremities. The injured rats were killed between 30 minutes and 14 days after injury, and the injured cord was removed en bloc. Rats that received NMDA receptor antagonist (MK-801) were killed at the same time points as those that received saline. The specimens were stained with hematoxylin and eosin, Nissl, and Klüver—Barrera Luxol fast blue and subjected to in situ nick-end labeling, a specific in situ method used to allow visualization of apoptosis. Thirty minutes post-SCI, a large hematoma was observed at the compressed segment. Six hours after injury, large numbers of dead cells that were not stained by in situ nick-end labeling were observed. Between 12 hours and 14 days postinjury, nuclei stained by using the in situ nick-end labeling technique were observed not only at the injury site but also in adjoining segments that had not undergone mechanical compression, suggesting that the delayed cell death was due to apoptosis. The number of cells stained by in situ nick-end labeling was maximum at 3 days postinjury. The results of electron microscopic examination were also consistent with apoptosis. In the MK-801—treated rats, the number of cells stained by in situ nick-end labeling was smaller than in nontreated rats at both 24 hours and 7 days after injury. Conclusions. These findings suggest that NMDA-receptor activation promotes delayed neuronal and glial cell death due to apoptosis.