Morphine blood levels, dependence, and regulation of hippocampal subgranular zone proliferation rely on administration paradigm.

Chronic morphine, administered via s.c. pellet, decreases the number of proliferating cells in the dentate gyrus subgranular zone (SGZ) in both rats and mice. This robust morphine-induced decrease could be used to better understand mechanisms regulating adult hippocampal neurogenesis, as well as to explore the relationship between neurogenesis and drug dependence, withdrawal, and relapse behaviors. Such research would benefit enormously from identifying a route of morphine administration that produces addiction-relevant blood levels of morphine, results in a high degree of dependence, translates to both rat and mouse, and is free of the behavioral confounds of s.c. pellets. Therefore, we examined a classic chronic morphine pellet paradigm (two s.c. pellets over 5 days) versus three chronic morphine injection paradigms (escalating dose i.p. injections over 2, 5, or 10 days) for their effect in adult male C57BL/6J mice. We assessed blood morphine levels, SGZ proliferation, and drug dependence as assessed by tolerance to locomotion sensitization and naloxone-precipitated withdrawal. The pellet paradigm produced high and relatively stable blood levels of morphine, a high degree of dependence, and a significant decrease in SGZ proliferation. In contrast, the three injection paradigms produced transient spikes in morphine blood levels, significantly less dependence than the pellet paradigm, and no significant decrease in SGZ proliferation. These data show that regulation of mouse SGZ proliferation requires high and relatively stable blood levels of morphine, and provide critical knowledge for the design of future studies to probe the relationship between addiction and neurogenesis.

Nerve growth factor rescue of cisplatin neurotoxicity is mediated through the high affinity receptor: studies in PC12 cells and p75 null mouse dorsal root ganglia.

Nerve growth factor (NGF) rescues dorsal root ganglion neurons and PC12 cells from cisplatin-induced cell death. Two model systems were used to demonstrate that rescue is mediated through the high affinity NGF receptor. In dorsal root ganglion (DRG) neurons isolated from p75(-/-) and control mice, 20 ng/ml NGF completely prevented cisplatin-induced death. In PC12 cells, we overexpressed receptor chimeras between the tumor necrosis factor and NGF receptors. We demonstrated that activation of the intracellular domain of Trk A is responsible for the NGF rescue effect.

Effect of chronic morphine on the dentate gyrus neurogenic microenvironment.

Opiates, such as morphine, decrease neurogenesis in the postnatal hippocampal subgranular zone (SGZ) by inhibiting progenitor proliferation and maturation. However, it is not known how morphine influences the growth factors and vasculature that encompass the neurogenic SGZ microenvironment. We examined morphine's effect on pro- and anti-proliferative factors in the dentate gyrus (DG; Experiment 1) as well as the DG neurovasculature itself (Experiment 2). For Experiment 1, mice were implanted with subcutaneous sham or morphine pellets (0 and 48 h) and were decapitated 24 or 96 h later. One brain hemisphere was postfixed to examine proliferation by immunohistochemistry, and a DG-enriched sample was dissected from the other hemisphere to examine the neurogenic microenvironment via immunoblotting for known pro- and anti-proliferative factors. Consistent with previous results, morphine decreased the number of proliferating cells in the SGZ, as the number of Ki67-immunoreactive (IR) cells was decreased at 96 h. Morphine did not alter DG levels of the pro-proliferative factor brain-derived neurotrophic factor, anti-proliferative factor interleukin-1 beta, or their receptors TrkB and IL1R1 at either time point. However, morphine increased the pro-proliferative factor vascular endothelial growth factor (VEGF) at 96 h. Given that VEGF is also a potent angiogenic factor, Experiment 2 examined whether the morphine-induced increase in VEGF correlated with altered DG neurovasculature. Mice were implanted with morphine pellets as in Experiment 1, and 2 h before perfusion (24 or 96 h) were administered bromodeoxyuridine (BrdU; intraperitoneal, 150 mg/kg). Tissue was co-stained for BrdU and the endothelial cell marker endoglin to enable examination of DG vessels and proximity of BrdU-IR cells to endoglin-IR vessels. At 96 h, endoglin-IR vessel area and perimeter were increased, but proximity of BrdU-IR cells to endoglin-IR vessels remained unchanged. These data suggest that following chronic morphine exposure, factors within the neurogenic microenvironment are maintained or upregulated to compensate for decreased SGZ proliferation.

The medial triplane fracture: report of an unusual injury.

A case report of a medial triplane fracture of the distal tibia is presented, with successful open reduction of the fracture. The injury occurs across three planes, with medial and posterior displacement. Anatomic reduction is necessary to prevent articular surface incongruity or a growth disturbance.

Inhibition of axonal growth from sensory neurons by excess nerve growth factor.

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NGF). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.

Alterations in cell cycle regulation underlie cisplatin induced apoptosis of dorsal root ganglion neurons in vivo.

Cisplatin is used in the treatment of ovarian and testicular cancer. Twenty percent of patients cannot be optimally treated because of sensory neurotoxicity. Human and animal studies demonstrate that the dorsal root ganglion neuron is the primary target of drug injury. We have previously demonstrated that cisplatin causes neuronal apoptosis in vitro. We now report a reproducible animal model of cell death induced by cisplatin. Drug was administered for 1 or 2 cycles of 5 days separated by 5 days. Total dose administered was 0, 5, 7.5, 10, or 15 mg/kg. Ganglia from 34 animals were processed and examined using in situ hybridization for cyclin D1 messenger RNA and digoxigenin coupled TUNEL staining. Overall, 2.9 +/- 3.9% of neurons were TUNEL positive in treated rats compared with 0.2 +/- 0.3% in controls (P <.005). There was a strong positive correlation (r2 = 0.88; P = 0.018) between percentage of TUNEL stained DRG and cumulative dose of cisplatin. Two independent approaches to quantitation of in situ cyclin D1 hybridization were used; blinded grading by an observer and measurement of color density using digital image analysis. Both demonstrated dramatic upregulation of expression of cyclin D1 mRNA in treated compared with control rats. This demonstrates that apoptosis of neurons is preceded by aberrant reentry into G1 phase of the cell cycle in an animal model.