Jamil Ahsan Kazi

Involvement of Oxidative Stress in Tumor Cytotoxic Activity of Hepatocyte Growth Factor/Scatter Factor

In this study, we show thatN-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin trapsN-t-butyl-α-phenylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2′,7′-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.

Gabapentin completely attenuated the acute morphine induced c-Fos expression in the rat striatum

The neuro-anatomical sites and molecular mechanism of action of gabapentin (GBP)-morphine interaction to prevent and reverse morphine side effects as well as enhancement of the analgesic effect of morphine is not known. Therefore, we examined the combined effects of GBP-Morphine on acute morphine induced c-Fos expression in rat striatum. The combined effect of GBP-Morphine was examined by means of c-Fos immunohistochemistry. A single intraperitoneal injection (i.p.) of morphine (10 mg/kg), saline (control), co-injection of GBP (150 mg/kg) with morphine (10 mg/kg) was administered under anaesthesia. Ninety minutes after drugs administration the deeply anesthetized rats were perfused transcardially with 4% paraformaldehyde. Serial 40 µm thick sections of brain were cut and processed by immunohistochemistry to locate and quantify the sites and number of neurons with c-Fos immunoreactivity. Detection of c-Fos protein was performed using the peroxidase-antiperoxidase (PAP) detection protocol. Our present study demonstrated that, administration of GBP (150 mg/kg, i.p.) in combination with morphine (10 mg/kg, i.p.) significantly (p<0.01) attenuated the acute morphine (10 mg/kg, i.p.) induced c-Fos expression in the rat striatum. Present results showed that GBP-morphine combination action prevented the acute morphine induced c-Fos expression in rat striatum. Moreover, this study provides first evidence of neuro-anatomical site and that GBP neutralized the morphine induced activation of rat striatum.

Prolonged expression of c-Fos protein in the lateral habenular nucleus of the Japanese monkey (Macaca fuscata) after eye enucleation.

To elucidate the effect of traumatic stress on the lateral habenular nucleus, we investigated the time course of the expression of c-Fos protein in this nucleus of the Japanese monkey (Macaca fuscata) after enucleation of one eye using c-Fos protein immunocytochemistry. c-Fos protein-like immunoreactive neurons were significantly increased; the increase started 1 h after the enucleation and remained high for 3–9 h in the lateral habenular nucleus on both sides. These results suggest that the prolonged expression of c-Fos protein occurred in the lateral habenular nucleus after traumatic stress through multiple transsynaptic activations.

Localization of nocistatin-binding sites in mice brain and spinal cord using a biotinylated nocistatin probe.

Nocistatin and nociceptin/orphanin FQ are two neuropeptides processed from the same precursor prepronociceptin. They have opposing roles in nociception and several other biological functions. Whereas the location and structure of the nociceptin/orphanin FQ receptors has been defined, the location of the nocistatin receptors remains unknown. In the course of this study, we synthesized a novel probe for histochemistry by linking biotin to the N terminus of nocistatin, and purified this with high-pressure liquid chromatography and confirmed the structure by mass spectrometer. Using this probe, we found nocistatin-binding sites in the cerebral cortex and the dorsal horn nucleus of the spinal cord. We also found that the nocistatin-binding sites were in the cell body, whereas the nociceptin/orphanin FQ binding sites were on the fibrous processes.

Nocistatin attenuated the nociceptin induced c-Fos expression in the mouse hippocampus

Nocistatin and nociceptin/orphaninFQ (N/OFQ) are the two new peptides which may have roles in nociception, memory, anxiety, and other biological functions. Nocistatin acts as a functional antagonist to N/OFQ in several functions, but their neuro-anatomical sites of interaction are unknown. We investigated the effect of combined intracerebroventricular (i.c.v.) injection of nocistatin with N/OFQ, on N/OFQ induced c-Fos expression in the mouse hippocampus, using c-Fos immunohistochemistry. We found that co-injection of nocistatin with N/OFQ significantly attenuated N/OFQ induced c-Fos expression in the hippocampus.

Effect of enucleation on the expression of c-Fos protein in the supraoptic nucleus of the Japanese monkey (Macaca fuscata)

The aim of this study was to examine the effects of one eye enucleation on the expression of c-Fos protein in the hypothalamus of the Japanese monkey (Macaca fuscata). Compared with an intact monkey, significantly increased numbers of c-Fos positive neurons were observed in the supraoptic nuclei on both sides at 1 h after eye enucleation. This maximal c-Fos expression then started to decrease at 3 h after eye enucleation. Furthermore, by a dual-labeled immunocytochemical study, the c-Fos immunoreactivity was found mainly in the vasopressinergic but not in the oxytocinergic neurons within the supraoptic nucleus. These results suggest that vasopressinergic but not oxytocinergic neurons within the supraoptic nucleus may have critical roles in the stimulation of this nucleus in response to eye enucleation.

Nocistatin and nociceptin modulate c-Fos expression in the mice thalamus

Nocistatin and nociceptin/orphanin FQ (N/OFQ) are two neuropeptides which may have opposite effects in several biological functions but their neuro-anatomical sites of interaction are not fully clear. We investigated interaction between the effect of intracerebroventricular (i.c.v.) injection of nocistatin and N/OFQ, on c-Fos expression in the mouse thalamus, using c-Fos immunohistochemistry. We found that co-injection of nocistatin with N/OFQ significantly modulates c-Fos expression in the thalamus. The present study strongly suggests that “Nocistatin–Nociceptin” interaction system in the thalamus may be the promising neuromodulatory sites in the investigation of unlocking their possible therapeutic circuit in nociception, memory and anxiety.

Gabapentin Differentially Modulate c-Fos Expression in Hypothalamus and Spinal Trigeminal Nucleus in Surgical Molar Extraction

The study on the efficacy of oral analgesics reported that no single class of drug is effective in post-surgical dental pain. Pain following removal of third molar is most commonly used and widely accepted acute pain model for assessing the analgesic effect of drugs in humans. Reports demonstrated that analgesic efficacy in the human dental model is highly predictive. The high incidence of false-negative findings in analgesic investigations hinders the process of molecular discovery. Molecular mechanism of post-surgical pain is not known. More importantly, the animal model for postoperative dental pain is not well established. In an attempt to discover an effective post-surgical dental pain blocker with acceptable side effects, it is essential to elucidate the molecular mechanism of post-operative dental pain. The present study investigated mandibular molars extraction in rat as an animal model for the post-operative dental pain in central nervous system. Using c-Fos immunohistochemistry, we demonstrated that pre administration of GBP (150 mg/kg. i.p) significantly (p< 0.01) neutralized the surgical molar extraction induced c-Fos expression bilaterally in rat hypothalamus. Present results indicate that pain after surgical molar extraction might follow novel neural pathways therefore difficult to treat with existing anti-nociceptive drugs.