Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a model for nitric oxide in major depressive disorder.

Nitric oxide (NO) and NO synthase-1 (NOS1) are involved in the stress response and in depression. We compared NOS-NO alterations in rats exposed to chronic unpredictable stress (CUS) with alterations in major depressive disorder (MDD) in humans. In the hypothalamus of male CUS rats we determined NOS activity, and in the paraventricular nucleus (PVN) we determined NOS1-immunoreactive (ir) cell densities and co-localization of NOS1 with stress-related neuropeptides corticotropin-releasing hormone (CRH), vasopressin (AVP) or oxytocin (OXT). We measured plasma NO levels and cortisol in male medicine-naïve MDD patients and plasma NO and corticosterone (CORT) in CUS rats. In the CUS rat total NOS activity in the hypothalamus (P=0.018) and NOS1-ir cell density in the PVN were both significantly decreased (P=0.018), while NOS1 staining was mainly expressed in OXT-ir neurons in this nucleus. Interestingly, plasma NO levels were significantly increased both in male CUS rats (P=0.001) and in male MDD patients (P<0.001). Plasma CORT levels were increased in male CUS rats (P=0.001), while male MDD patients did not show a significant change in cortisol levels. In conclusion, the changes in plasma and hypothalamic NOS-NO of CUS rats and MDD were similar. The male CUS rat model may thus help us with our investigation of the mechanism underlying NOS-NO alterations in depression.

Reduced GAD(65/67) immunoreactivity in the hypothalamic paraventricular nucleus in depression: a postmortem study.

BACKGROUND: Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter. It diminishes the activity of the hypothalamo-pituitary-adrenal (HPA) axis, which plays an important role in the pathogenesis of depression. The present study aimed at determining GABAergic input in the hypothalamic paraventricular nucleus (PVN) in depression and its correlation with the activity of corticotropin-releasing hormone (CRH) neurons. METHODS: The density of glutamic acid decarboxylase (GAD)(65/67)-immunoreactivity (ir) was quantified in the postmortem hypothalamic PVN of 9 major depressive (MDD) and 5 bipolar depressive (BD) patients, together with 12 matched controls, whose CRH-expressing neuron numbers had been determined in a previous study. RESULTS: There was a 43% significant reduction of the density of GAD(65/67)-ir in the PVN in MDD (P=0.028) and a 20% non-significant decrease in BD patients. In addition, there was a significant negative correlation between the density of GAD(65/67)-ir and the number of CRH-ir neurons in the PVN in the depression group (Rho=-0.527, P=0.032), but not in the control group. LIMITATIONS: The samples were relatively small and the depression group had used antidepressants. CONCLUSION: A diminished GABAergic input to the PVN may contribute to the activation of CRH-ir neurons in depression, most prominently in MDD, which provides a rationale for prescribing GABAergic agonists for these patients.

Decreased NOS1 expression in the anterior cingulate cortex in depression.

Decreased function of the anterior cingulate cortex (ACC) is crucially involved in the pathogenesis of depression. A key role of nitric oxide (NO) has also been proposed. We aimed to determine the NO content in the cerebrospinal fluid (CSF) and the expression of NO synthase (NOS) isoforms, that is, NOS1, NOS2, and NOS3 in the ACC in depression. In depressive patients, CSF-NOx levels (the levels of the NO metabolites nitrite and nitrate) were significantly decreased (P = 0.007), indicating a more general decrease of NO production in this disorder. This agreed with a trend toward lower NOS1-mRNA levels (P = 0.083) and a significant decrease of NOS1-immunoreactivity (ir) (P = 0.043) in ACC. In controls, there was a significant positive correlation between ACC-NOS1-ir cell densities and their CSF-NOx levels. Furthermore, both localization of NOS1 in pyramidal neurons that are known to be glutamatergic and co-localization between NOS1 and GABAergic neurons were observed in human ACC. The diminished ACC-NOS1 expression and decreased CSF-NOx levels may be involved in the alterations of ACC activity in depression, possibly by affecting glutamatergic and GABAergic neurotransmission.

[Maternal deprivation-induced stress responses in female rats].

OBJECTIVE: To investigate the effect of maternal deprivation on the activity of hypothalamo-pituitary-adrenal (HPA) axis, acute stress response and the sex hormone receptors expression in hypothalamic paraventricular nucleus (PVN) in female rats. METHODS: Maternal deprivation model was induced in female Sprague-Dawley (SD) rats. Foot shock was given at different stages of estrus cycle during the adulthood. Plasma estradiol, testosterone and adrenocorticotropin (ACTH) levels were determined by radioimmunoassay; and plasma corticosterone level was measured by enzyme linked immunosorbent assay. The expression of androgen receptor (AR) and estrogen receptor (ER-ß) in the hypothalamic PVN was detected by immunohistochemistry. RESULTS: Decreased plasma ACTH and corticosterone levels were found in the proestrus of female rats with maternal deprivation (P=0.012 and P=0.019, respectively). A significant down-regulation (P=0.008) of PVN-AR, but not PVN-ER-ß expression was found in female rats with maternal deprivation. CONCLUSION: Maternal deprivation may reduce the HPA axis activity in female SD rats, which is closely correlated with the fluctuation of the circulating sex hormones. The androgen in the hypothalamus seems to play a more important role than the estrogen in this procedure.

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy.

BACKGROUND AND OBJECTIVE: Vaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy. DESIGN AND SETTING: Animal study conducted in the First Affiliated Hospital of Xi'an Jiaotong University from January 2009 to January 2010. PATIENTS AND METHODS: Lymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (T(E)). T(E) were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated. RESULTS: Compared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups. CONCLUSIONS: Lymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

Corticotropin-releasing hormone, glutamate, and γ-aminobutyric acid in depression.

Stress response and depression have a significant impact on modern society. Although the symptoms are well characterized, the molecular mechanisms underlying depression are largely unknown. The monoamine hypothesis, which postulates dysfunctional noradrenergic and serotonergic systems as the underlying primary cause of depression, has been valuable for the development of conventional antidepressants, which can reverse these dysfunctional states to some degree. However, recent data from various neuroscience disciplines have questioned the major role of amines in the pathogenesis of depression. A considerable amount of evidence has accumulated that suggests that normalization of the hypothalamo-pituitary-adrenal (HPA) system might be the final step necessary for a remission of depression. In addition, an increasing body of clinical and postmortem evidence is pointing to a role played by γ-aminobutyric acid (GABA) and glutamate in the etiology of depression. This review examines the evidence, mainly obtained from clinical studies or from postmortem brain material, for a major role of the HPA axis, glutamatergic, and GABAergic systems in the pathogenesis of major and bipolar depression. The authors hope that these insights will stimulate further studies with the final aim of developing new types of antidepressants that combine increased efficacy with a shorter delay of the onset of action and reduced side-effect profiles.

[Evaluation of immune reconstitution with in vitro-activated T cells from a tumor-bearing host].

AIM: To evaluate the reconstituted immune system with in vitro-activated T cells from tumor-bearing rats coupled with ovarian cancer vaccination. METHODS: Fischer 344 female rats were injected with cyclophosphamide (CY) as a lymphopenia (LP) model. The immune systems of the rats were reconstituted with in vitro-activated T cells from the same individuals. GM-CSF-modified ovarian cancer cells lines (NuTu-19) were injected within 24 h after immune reconstitution. The tumor vaccine draining lymph nodes (TVDLN) were harvested 8-10 days after vaccination and analyzed by FACS. The proliferative capacity of dendritic cells (DCs) was measured by the levels of MHC-II and CD86 molecules. The activation of T cells was monitored by the percentage of FITC-CD4 and PE-CD8 cells. The biological function of DCs such as processing and presenting antigens was assayed by immature DCs' phagocytosis of FITC-Dextran. RESULTS: Immune reconstitution with in vitro-activated T cells produced significantly more DCs, T cells and functionally enhanced immature DCs out of TVDLN. CONCLUSION: Reconstituting immune system with in vitro-activated T cells from a tumor-bearing host coupled with ovarian cancer vaccination during lymphocytopenia may selectively expand and activate particular T cells and DCs, leading to augmentation of anti-tumor immunity.

[Animal experiments of immunotherapy with ovarian tumor vaccine during lymphopenia induced by chemotherapy.].

OBJECTIVE: To explore the mechanisms and effects of adoptive immunotherapy with ovarian cancer vaccine modified by GM-CSF gene which was used after immunologic reconstitution during lymphopenia induced by chemotherapy. METHODS: Lymphopenia was induced by chemotherapy with cyclophosphamide. The immune reconstituted model was built in rats. The tumor vaccine draining lymph nodes were harvested after the ovarian cancer cells NUTU-19 modified by GM-CSF gene were injected. The effector T cells (T(E)) were got after being stimulated and amplified. Enzyme-linked immunosorbent assay was used to detect the level of interleukin (IL)-2 and IL-4 secreted by T(E). Intracellular cytokine staining was used to determine frequency of tumor-specific T(E). Fluorescence-activated cell sorting (FACS) was used to detect the special cytotoxicity of T(E) killing target cells. The survival period of rats bearing pre-established abdominal ovariam carcinoma after being adoptively transferred by T(E) was observed. RESULTS: Compared with those in control group, the significant higher levels IL-2 [(65.7 +/- 4.0) pg/ml] and lower levels IL-4 [(277 +/- 49) pg/ml] were observed in chemotherapy-immune reconstitution-vaccine immunization group. The amount of CD(4)(+) T cells secreting interferon-gamma (13.0 +/- 2.1)% were also significantly increased. The rate of the special cytotoxicity of killing T cells (86.5 +/- 1.1)% was markedly improved. The survival period of rats (110 +/- 16) days was increased in chemotherapy-immune reconstitution-vaccine immunization group. CONCLUSIONS: The combined immunotherapy of chemotherapy-immune reconstitution-tumor vaccine immunotherapy may increase the frequency and function of specific tumor T(E). The specific cytotoxicity is increased and the weak reaction of T(E) to tumor is improved, which showed that this therapy can enhance immune reaction.

Altered gene expression of NIDD in dorsal root ganglia and spinal cord of rats with neuropathic or inflammatory pain.

Nitric oxide and nitric oxide synthases are key players in synaptic plasticity events in spinal cord (SC), which underlies the chronic pain states. To date, little is known about the molecular mechanisms regulating the activity of nitric oxide synthases in nociceptive systems. The present study was aimed at the determination of the gene expression of nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD), a recently identified protein regulating nNOS enzyme activity, in rat SC and dorsal root ganglia (DRG) and studying its regulation in states of nociceptive hypersensitivity in a rat model of neuropathic or inflammatory pain. It was found that NIDD mRNA was predominantly expressed in nociceptive primary neurons and in neurons of the spinal dorsal horn (DH) and the number of NIDD-positive neurons in the corresponding DRG or SC increased significantly following induction of chronic hyperalgesia. Meanwhile, remarkable changes of nNOS were detected under such pain conditions. Our data suggest a potential role for NIDD in the maintenance of thermal pain hypersensitivity possibly via regulating the nNOS activity.

Dynamic changes of p27(kip1) and Skp2 expression in injured rat sciatic nerve.

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27(kip1). Previous reports have showed that p27(kip1 )played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27(kip1 )and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27(kip1). By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27(kip1) levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27(kip1 )and Skp2 likely play an important role in peripheral nerve injury and regeneration.