TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.

TNF-alpha is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-alpha in the bony repair of injured growth plate were examined in young rats treated with a TNF-alpha antagonist. The results show that TNF-alpha mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate. INTRODUCTION: TNF-alpha inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-alpha signaling is essential for bone fracture healing. Roles of TNF-alpha in the bony repair of injured growth plate cartilage are unknown. MATERIALS AND METHODS: Roles of TNF-alpha in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-alpha inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-alpha and IL-1beta, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-alpha signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-alpha, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs. RESULTS: p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-alpha antagonist, suggesting a role of TNF-alpha in p38 activation. Whereas TNF-alpha inhibition did not alter inflammatory infiltrate and expression of TNF-alpha and IL-1beta at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-alpha increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-alpha inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-alpha and cbfa1 expression levels, suggesting a negative relationship between TNF-alpha and cbfa1 in this in vivo model. CONCLUSIONS: These observations suggest that TNF-alpha activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-alpha-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site.     

Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.

Salter's type III and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibial transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-X immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-alpha1) on day 3, along with some cbf-alpha1+ osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphatase-immunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.     

Neurotrophin‐3 Induces BMP‐2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill‐hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT‐3, and NT‐4 and their Trk receptors. NT‐3 and its receptor TrkC showed the highest induction. NT‐3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT‐3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT‐3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP‐2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT‐3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP‐2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT‐3 osteogenic effect in vitro because it can be almost completely abrogated by co‐addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT‐3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co‐treatment with anti‐VEGF. This study suggests that NT‐3 may be an osteogenic and angiogenic factor upstream of BMP‐2 and VEGF in bony repair, and further studies are required to investigate whether NT‐3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.     

Roles of Wnt/β-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats

Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt–β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt–β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200 mg/kg/day for 8 days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n = 8, P < 0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P < 0.01). At 25 mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.     

Temporal and spatial expression of transforming growth factor-β in the healing patellar ligament of the rat

We investigated the temporal and spatial expression of transforming growth factor-β in the healing patellar ligament of the rat by immunohistochemistry. The mid-portion of the medial half of the patellar ligament in 14-week-old male Wistar rats was cut transversely with a scalpel. On day 1 after ligament injury, diffuse staining for transforming growth factor-β was observed in the extracellular matrix filling the wound, and the staining in the adjacent ligament tissue was as weak as it was in the normal ligament. On day 3, the intensity of the diffuse extracellular staining decreased, and the staining was observed in correspondence with the cellular distribution in the wound site and in the adjacent uninjured ligament tissue. On day 7, the intense staining was widely distributed over the whole length of the ligament tissue. On day 28, the staining for transforming growth factor-β was still observed at the wound site and in the adjacent uninjured ligament tissue, where the staining was reduced in intensity but still stronger than it was in the normal ligament. On day 56, the expression of transforming growth factor-β was still detectable at the wound site: however, in the adjacent uninjured ligament tissue, it had almost subsided to the normal level. The results of the present study suggest that ligament healing may be accompanied by extensive changes in the expression of transforming growth factor-β over the whole length of ligament tissue.     

Tibiofemoral bony morphology features associated with ACL injury and sex utilizing three-dimensional statistical shape modeling

Statistical shape modeling was employed to assess three-dimensional (3D) bony morphology between distal femurs and proximal tibiae of anterior cruciate ligament (ACL) injured knees, the contralateral uninjured knees of ACL injured subjects, and knees with no history of injury. Surface models were created by segmenting bone from bilateral computed-tomography scans of 20 subjects of their ACL injured knees and non-injured contralateral knees, and 20 knees of control subjects with no history of a knee injury. Correspondence particles were placed on each surface, and a principal component analysis determined modes of variation in the positions of the correspondence particles describing anatomical variation. ANOVAs assessed the statistical differences of 3D bony morphological features with main effects of injury state and sex. ACL injured knees were determined to have a more lateral femoral mechanical axis and a greater angle between the long axis and condylar axis of the femur. A smaller anterior–posterior dimension of the lateral tibial plateau was also associated with ACL injured knees. Results of this study demonstrate that there are more bony morphological features predisposing individuals for ACL injury than previously established. These bony morphological parameters may cause greater internal and valgus torques increasing stresses in the ACL. No differences were determined between the ACL injured knees and their uninjured contralateral knees demonstrating that knees of ACL injured individuals are at similar risk for injury. Further understanding of the effect of bony morphology on the risk for ACL injury could improve individualized ACL injury treatment and prevention.     

ADAM12-S stimulates bone growth in transgenic mice by modulating chondrocyte proliferation and maturation.

ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell-extracellular matrix interactions in the growth plate. INTRODUCTION: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported. MATERIALS AND METHODS: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell-extracellular matrix interactions. RESULTS: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced in mice expressing higher levels of the transgene than in a lower-expressing line. Histological analysis revealed no alterations in the growth plate organization, but mean growth plate width was increased. Both the cellular incorporation of bromodeoxyuridine and the width of the collagen type X-positive hypertrophic zone were increased in the growth plate of ADAM12-S transgenic mice. Importantly, mice expressing a truncated form of ADAM12-S that lacked the pro- and metalloprotease domains showed no alterations in bone length, suggesting that protease activity is required for the ADAM12-S effect. In vitro studies showed that ADAM12-S inhibits chondrocyte adhesion to fibronectin and collagen type II. CONCLUSIONS: ADAM12-S stimulates bone growth in mice by modulating chondrocyte proliferation and maturation through mechanisms probably involving both metalloprotease and adhesion activities.     

Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats

With the intensified use of chemotherapy and improved survival rates for childhood malignancies, it has become increasingly apparent that some children or adult survivors show poor bone growth and develop osteoporosis. As a step to investigate underlying mechanisms, this project examined short-term effects in rats of chemotherapy agent 5-fluorouracil (5-FU) on cell proliferation, apoptosis, and bone formation at tibial growth plate cartilage and its adjacent bone-forming region metaphysis. In addition, since insulin-like growth factor (IGF-I) is important for bone growth, we examined whether IGF-I pretreatment would potentially protect growth plate cartilage and bone cells from chemotherapy damage. Two days after a single high dose of 5-FU injection, proliferation of growth plate chondrocytes and metaphyseal osteoblasts/preosteoblasts was dramatically suppressed, and apoptosis was induced among osteoblasts and preosteoblasts. As a result, there was a reduction in the chondrocyte number and zonal height at the proliferative zone and a decline in the number of osteoblasts and preosteoblasts on the metaphyseal trabecular bone surface. At day 2, no obvious deleterious effects were observed on the height of the growth plate hypertrophic zone and the bone volume fraction of the metaphyseal primary spongiosa trabeculae. At day 10, while cell proliferation and growth plate structure returned to normal, there were slight decreases in trabecular bone volume, body length increase, and tibial length. While pretreatment with 1-week IGF-I systemic infusion did not attenuate the suppressive effect of 5-FU on proliferation in both growth plate and metaphysis, it significantly diminished apoptotic induction in metaphysis. These results indicate that growth plate cartilage chondrocytes and metaphyseal bone cells are sensitive to chemotherapy drug 5-FU and that IGF-I pretreatment has some anti-apoptotic protective effects on metaphyseal bone osteoblasts and preosteoblasts.     

Changes in young rat radius following excision of the perichondrial ring

Summary The distal growth plate of the radius was exposed in young rats and the perichondrial ring and fibrous covers were removed at the exposed surface. In addition, a small portion of either the adjacent epiphysis or of the adjacent metaphysis was removed also. In the following days, important alterations in bone structure were observed at the level of the removed perichondrial ring. The most relevant changes were an enlargement of the growth plate at the exposed surface that grew in an abnormal direction, proliferation of bone trabecules at the level of the excised perichondrial ring, and bending of the bone. No regeneration of the perichondrial ring occurred. These changes support both the role of the perichondrial ring in the mechanical constraint of the growth plate, and the induction of bone formation by the hypertrophic cartilage at the level of the absent perichondrial ring.     

Cellular mechanisms for methotrexate chemotherapy-induced bone growth defects

Methotrexate (MTX) is a commonly used anti-metabolite in childhood oncology and is known to cause bone growth arrest and osteoporosis; yet the underlying mechanisms for MTX-induced bone growth defects remain largely unclear. This study characterized damaging effects in young rats of acute chemotherapy with 5 once-daily doses of MTX (0.75 mg/kg) on the cellular activities in the growth plate in producing calcified cartilage and trabecular bone and on activities of osteoblastic cells in the metaphysis. MTX treatment significantly induced chondrocyte apoptosis. MTX also suppressed chondrocyte proliferation and reduced collagen-II mRNA expression and total thickness of the growth plate, with the damage being most obvious on day 9 after the first injection, and with the growth plate histological structure returning normal on day 14. In the adjacent metaphyseal bone, mirroring the decrease in the width of the growth plate, production of primary spongiosa bone was markedly reduced and bone volume of the secondary spongiosa was decreased. Furthermore, MTX treatment significantly induced osteocyte apoptosis in the primary spongiosa and reduced proliferation of osteoblasts and preosteoblasts particularly in the secondary spongiosa. These observations suggest that methotrexate chemotherapy may cause bone growth defects by arresting cellular activities in the growth plate in producing calcified cartilage and primary trabecular bone and by decreasing pools of metaphyseal osteoblastic cells. However, this short-term MTX treatment only caused transit suppressions on growth plate cartilage and trabecular bone, as most cellular and histological parameters had recovered by day 14 or 21.     

骨骺损伤修复过程中骺板形态及VEGF表达的变化

 目的 探讨大鼠骨骺损伤修复过程中骺板形态结构及骺板内血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)表达的变化。方法 取4~5 周龄SD大鼠30 只, 制作胫骨近端骨骺损伤动物模型。随机分为五组, 每组6 只, 分别于术后2、4、6、10、21 天处死, 取双侧胫骨。测量双侧胫骨长度, 计算实验侧长度和对照侧长度比值。行双侧胫骨X 线摄片、Micro CT扫描, 观察骺板大体形态及骺板内骨桥形成情况。通过组织切片HE 染色及免疫组织化学染色, 观察骺板内软骨细胞变化及VEGF表达。结果 双侧肢体长度于术后第4 天出现差异, 第10 天差异最大, 第21 天肢体长度差异缩小。术后第6 天开始骺板内逐渐出现纤细骨质, 终至骨桥形成。HE 染色提示伤后早期出现静止区软骨细胞聚集成团, 细胞规律性排列丧失、分化加速。免疫组织化学染色显示术后生长板内出现VEGF 高反应区, 阳性表达区逐渐扩大, 跨越骺板全层, 直后血管长入、骨化。结论 骨骺损伤修复过程中, 骺板形态早中期表现为干骺端肥大, 肢体短缩, 骺板内纤细骨质形成;随修复进展, 骺板内VEGF 表达反应性增强, 出现贯通骺板的表达带, 与骨桥形成相关。     

Uptake and elimination of methylprednisolone from contused cat spinal cord following intravenous injection of the sodium succinate ester

The uptake and elimination of methylprednisolone by the injured cat lumbar spinal cord were examined following a single 30-mg/kg intravenous bolus injection of the sodium succinate ester. The findings were considerably different from those previously reported for normal lumbar cord. When the glucocorticoid was administered 30 minutes after a 400 gm-cm contusion injury, peak tissue concentrations in both injured and uninjured segments of traumatized spinal cord were not achieved until 30 minutes following drug administration. The elimination of methylprednisolone from injured spinal cord tissue was biphasic in nature, with a rapid elimination phase occurring between 1 and 2 hours after drug administration. This rapid elimination phase was followed by a slower phase which paralleled the constant elimination rate from uninjured tissue of traumatized cord (approximate half-time = 6 hours). Significantly more methylprednisolone accumulated in the injured segment of traumatized spinal cord than in an uninjured segment adjacent to the injury site. This was only true, however, if the drug was administered at times up to 1 hour after injury. If injected after 1 hour, uptake by the injured segment decreased significantly with time after trauma and was no different from that observed for the uninjured segment in the same animal, which showed no significant variation with time after trauma. The probable basis for these differences and the possible clinical implications of these pharmacokinetic characteristics are discussed.     

The effect of osteogenic protein-1 in an in vivo physeal injury model

The physis has limited ability to undergo repair, and injury may result in growth arrest. Osteogenic protein-1 promotes bone formation in diaphyseal defects, chondrocyte proliferation, and matrix synthesis. The authors' goal was to determine if the presence of osteogenic protein-1 in a defect involving the physis would promote cartilage repair, and in doing so, to determine the effect of osteogenic protein-1 on physeal growth. An ovine model of growth plate damage was used, in which the proximal medial physis of the tibia was partially ablated. The defect was filled with a Type I collagen paste containing osteogenic protein-1 (350 microg) or collagen alone. Growth rate was measured at 4, 14, and 56 days, and the defects were analyzed histologically at 4, 14, and 56 days. Bone bridge formation occurred within the defect site. However, osteogenic protein-1 promoted outgrowth of the adjacent physeal cartilage. The physeal cartilage underwent expansion until the mineral forming within the defect site blocked its progress. The effect was localized because only that portion of the physis at the defect margin appeared to be affected.     

Magnetic resonance imaging appearance of cartilage repair in the knee

Assessment of surgically repaired cartilage lesions with standardized cartilage sensitive magnetic resonance imaging was done to evaluate the integrity, morphologic features, and signal of the articular surface, thereby obtaining information about the natural history of these procedures in the knee. Magnetic resonance imaging also assessed the interface between the repaired and native cartilage, changes in the subchondral bone, and the appearance of cartilage over the opposite and adjacent (native) surfaces. One hundred eighty magnetic resonance imaging examinations were obtained in 112 patients who had cartilage-resurfacing procedures, including 86 microfractures and 35 autologous chondrocyte implantations, at a mean of 15 and 13 months after surgery, respectively. Autologous chondrocyte implantations showed consistently better fill of the defects at all times compared with microfracture. The graft hypertrophied in 63% of surgeries. The repair cartilage over the microfracture generally was depressed with respect to native cartilage. Propensity for bony overgrowth was most marked in the microfracture group, with loss of adjacent cartilage evident with progressive followup.     

Molybdenum-induced changes in the epiphyseal growth plate

Summary Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.     

Occult osseous lesions documented by magnetic resonance imaging associated with anterior cruciate ligament ruptures

Magnetic resonance images (MRI) were performed within three weeks of anterior cruciate ligament (ACL) rupture on 75 skeletally mature patients. Occult bony lesions were documented in 64 (85%) of the patients. Of the 64 patients with bone injuries, 83% had lesions of the lateral compartment. The lateral femoral condyle was involved in 50%, and the lateral tibial plateau was injured in 50% of the patients with changes. Nineteen of the 64 patients had more than one area of bony injury. Although the majority of bony lesions resolve, permanent alterations remained in some cases. This study has implications that may affect rehabilitation and the long-term prognosis in those patients with extensive bony and associated articular cartilage injuries.     

Traumatic brain injury and hemorrhagic hypotension suppress neuroprotective gene expression in injured hippocampal neurons

BACKGROUND: After traumatic brain injury, memory dysfunction is due in part to damage to the hippocampus. To study the molecular mechanisms of this selective vulnerability, the authors used laser capture microdissection of neurons stained with Fluoro-Jade to directly compare gene expression in injured (Fluoro-Jade-positive) and adjacent uninjured (Fluoro-Jade-negative) rat hippocampal neurons after traumatic brain injury and traumatic brain injury plus hemorrhagic hypotension. METHODS: Twelve isoflurane-anesthetized Sprague-Dawley rats underwent moderate (2.0 atm) fluid percussion traumatic brain injury followed by either normotension or hemorrhagic hypotension. Animals were killed 24 h after injury. Frozen brain sections were double stained with 1% cresyl violet and 0.001% Fluoro-Jade. RNA from 10 Fluoro-Jade-positive neurons and 10 Fluoro-Jade-negative neurons, obtained from the hippocampal CA1, CA3, and dentate gyrus subfields using laser capture microdissection, was linearly amplified and analyzed by quantitative ribonuclease protection assay for nine neuroprotective and apoptosis-related genes. RESULTS: In injured CA3 neurons, expression of the neuroprotective genes glutathione peroxidase 1, heme oxygenase 1, and brain-derived neurotrophic factor was significantly decreased compared with that of adjacent uninjured neurons. Superimposition of hemorrhagic hypotension was associated with down-regulation of neuroprotective genes in both injured and uninjured neurons of all subregions. Expression of apoptosis-related genes did not vary between injured and uninjured neurons, with or without superimposed hemorrhage. CONCLUSIONS: The authors show, in the first direct comparison of messenger RNA levels in injured and uninjured hippocampal neurons, that injured neurons express lower levels of neuroprotective genes than adjacent uninjured neurons.     

Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.

Skeletal growth depends on endochondral ossification in growth plate cartilage, where proliferation of chondrocytes, matrix synthesis, and increases in chondrocyte size all contribute to the final length of a bone. To learn more about the potential role of matrix synthesis/degradation dynamics in the determination of bone growth rate, we investigated the expression of matrix collagens and collagenase 3 in tibial growth plates in three age groups of rats (21, 35, and 80 days after birth), each characterized by specific growth rates. By combining stereological and in situ hybridization techniques, it was found that the expression of matrix collagens and collagenase 3 was specifically turned on or off at specific stages of the chondrocyte-differentiation cycle, and these changes occurred as a temporal sequence that varied depending of animal growth rate. Furthermore, the expression of these matrix proteins by a growth plate chondrocyte was found to be sped up or slowed down depending of the growth rate. In addition to expression of types II and X collagen, collagenase-3 expression was found to constitute a constant event in the series of changes in gene expression that takes place during the chondrocyte-differentiation process. Collagenase-3 expression was found to show a biphasic pattern: it was intermittently expressed at the proliferative phase and uniformly expressed at the hypertrophic stage. An intimate relationship between morphological and kinetic changes associated with chondrocyte hypertrophy and changes in the expression pattern of matrix collagens and collagenase 3 was observed. Present data prove that the matrix synthesis/degradation dynamics of the growth plate cartilage varied depending on growth rate; these results support the hypothesis that changes in matrix degradation and synthesis are a critical link in the sequence of tightly regulated events that lead to chondrocytic differentiation.