Jang Hern Lee

Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses

&NA; Bee venom (BV) has traditionally been used in Oriental medicine to relieve pain and to treat inflammatory diseases such as rheumatoid arthritis (RA). While several investigators have evaluated the anti‐inflammatory effect of BV treatment, the anti‐nociceptive effect of BV treatment on inflammatory pain has not been examined. Previous studies in experimental animals suggest that the therapeutic effect of BV on arthritis is dependent on the site of administration. Because of this potential site specificity, the present study was designed to evaluate the anti‐nociceptive effect of BV injections into a specific acupoint (Zusanli) compared to a non‐acupoint in an animal model of chronic arthritis. Subcutaneous BV treatment (1 mg/kg per day) was found to dramatically inhibit paw edema caused by Freunds adjuvant injection. Furthermore, BV therapy significantly reduced arthritis‐induced nociceptive behaviors (i.e. the nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). These anti‐nociceptive/anti‐inflammatory effects of BV were observed from 12 days through 21 days post‐BV treatment. In addition, BV treatment significantly suppressed adjuvant‐induced Fos expression in the lumbar spinal cord at 3 weeks post‐adjuvant injection. Finally, injection of BV into the Zusanli acupoint resulted in a significantly greater analgesic effect on arthritic pain as compared to BV injection in to a more distant non‐acupoint. The present study demonstrates that BV injection into the Zusanli acupoint has both anti‐inflammatory and anti‐nociceptive effects on Freunds adjuvant‐induced arthritis in rats. These findings raise the possibility that BV acupuncture may be a promising alternative medicine therapy for the long‐term treatment of rheumatoid arthritis.

The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats.

We recently demonstrated that bee venom (BV) injection into the Zusanli acupoint produced a significantly more potent anti-inflammatory and antinociceptive effect than injection into a non-acupoint in a Freunds adjuvant induced rheumatoid arthritis (RA) model. However, the precise BV constituents responsible for these antinociceptive and/or anti-inflammatory effects are not fully understood. In order to investigate the possible role of the soluble fraction of BV in producing the anti-arthritic actions of BV acupuncture, whole BV was extracted into two fractions according to solubility (a water soluble fraction, BVA and an ethylacetate soluble fraction, BVE) and the BVA fraction was further tested. Subcutaneous BVA injection (0.9 mg/kg/day) into the Zusanli acupoint was found to dramatically inhibit paw edema and radiological change (i.e. new bone proliferation and soft tissue swelling) caused by Freunds adjuvant injection. BVA treatment also reduced the increase in serum interleukin-6 caused by RA induction to levels observed in non-arthritic animals. In addition, BVA therapy significantly reduced arthritis-induced nociceptive behaviors (i.e. nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). Finally, BVA treatment significantly suppressed adjuvant-induced Fos expression in the lumbar spinal cord at 3 weeks post-adjuvant injection. In contrast, BVE treatment (0.05 mg/kg/day) failed to show any anti-inflammatory or antinociceptive effects on RA. The results of the present study demonstrate that BVA is the effective fraction of whole BV responsible for the antinociception and anti-inflammatory effects of BV acupuncture treatment. Thus it is recommended that this fraction of BV be used for long-term treatment of RA-induced pain and inflammation. However, further study is necessary to clarify which constituents of the BVA fraction are directly responsible for these anti-arthritis effects.

Intrathecal injection of the ς1 receptor antagonist BD1047 blocks both mechanical allodynia and increases in spinal NR1 expression during the induction phase of rodent neuropathic pain

Background: Selective blockade of spinal &sfgr;1 receptors (Sig-1R) suppresses nociceptive behaviors in the mouse formalin test. The current study was designed to verify whether intrathecal Sig-1R antagonists can also suppress chronic neuropathic pain. Methods: Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. The Sig-1R antagonist BD1047 was administered intrathecally twice daily from postoperative days 0 to 5 (induction phase of neuropathic pain) or from days 15 to 20 (maintenance phase). Western blot and immunohistochemistry were performed to determine changes in Sig-1R expression and to examine the effect of BD1047 on N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation in spinal cord dorsal horn from neuropathic rats. Results: BD1047 administered on postoperative days 0–5 significantly attenuated CCI-induced mechanical allodynia, but not thermal hyperalgesia, and this suppression was blocked by intrathecal administration of the Sig-1R agonist PRE084. In contrast, BD1047 treatment during the maintenance phase of neuropathic pain had no effect on mechanical allodynia. Sig-1R expression significantly increased in the ipsilateral spinal cord dorsal horn from days 1 to 3 after CCI. Importantly, BD1047 (30 nmol) administered intrathecally during the induction, but not the maintenance phase, blocked the CCI-induced increase in N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation. Conclusions: These results demonstrate that spinal Sig-1Rs play a critical role in both the induction of mechanical allodynia and the activation of spinal N-methyl-d-aspartate receptors in CCI rats and suggest a potential therapeutic role for the use of Sig-1R antagonists in the clinical management of neuropathic pain.

Nitric oxide synthase is found in some spinothalamic neurons and in neuronal processes that appose spinal neurons that express Fos induced by noxious stimulation

To determine if nitric oxide (NO) and Fos immunoreactivity induced by noxious stimulation were colocalized in spinothalamic neurons, double-staining immunocytochemical techniques were combined with retrograde neuroanatomical tracing procedures. Initial studies on three rats demonstrated that Fos and nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, did not coexist in spinothalamic tract neurons. However, some spinothalamic neurons were found to contain NOS and some NOS immunoreactive processes were found to appose Fos containing neurons. Thus the remainder of the study: (1) analyzed the relationship of NOS positive neuronal processes with Fos stained neurons using a Fos immunocytochemical technique in combination with either NOS immunofluorescence or NADPH-diaphorase histochemistry; and (2) quantitated the number of NOS containing cells that project to the thalamus using a combined immunofluorescent-retrograde tracing procedure. Both NOS-like immunoreactive (NOS IR) neuronal processes and NADPH-diaphorase positive neuronal processes in the dorsal horn of the lumbar spinal cord were found to appose Fos positive neurons located in laminae I and II of the dorsal horn. Approximately 40% of Fos-labeled cells in these superficial laminae were found to be in apposition to or in close proximity to NOS labeled neuronal processes. Examination of spinal cord sections for NOS-containing spinothalamic tract neurons revealed that lamina X was the only spinal cord region containing such double-labeled neurons. Further quantification revealed that approximately 10% of NOS positive neurons in lamina X were double-labeled with Fluorogold. These findings support the hypothesis that nitric oxide is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus and further indicate that nitric oxide may contribute to the central transmission of spinothalamic information.

Electroacupuncture modifies the expression of c-fos in the spinal cord induced by noxious stimulation

The present study was designed to investigate the effect of 4 Hz vs. 100 Hz electroacupuncture (EA) on c-fos expression in the spinal cord induced by noxious stimulation (NS). A second objective was to evaluate the sensitivity of these two different frequencies of EA stimulation to the opiate antagonist, naloxone. Mechanical NS was applied to the right hindpaw following 30 min of either 4 Hz or 100 Hz EA treatment and the resulting c-fos expression in the spinal cord dorsal horn was compared to that obtained in rats exposed only to the noxious stimulation. The involvement of endogenous opioids in the EA response to 4 Hz or 100 Hz stimulation frequencies was evaluated by pretreating rats with naloxone (5 mg/kg, i.p.) 10 min prior to EA. Both 4 Hz and 100 Hz EA reduced the number of c-fos-immunoreactive neurons in the spinal dorsal horn induced by noxious stimulation by 58% and 50%, respectively. The suppression of c-fos expression induced by 4 Hz EA was completely reversed by prior treatment with naloxone. On the other hand, the suppression of c-fos induced by 100 Hz EA was only partially blocked by this opiate antagonist. These data indicate that both high- and low-frequency EA are capable of inhibiting the expression of c-fos in the dorsal horn induced by NS. Low-frequency EA appears to be mediated primarily by endogenous opioid systems, while non-opioid mechanisms may be involved in mediating the analgesic effect of high frequency EA. These results support the hypothesis that EA has a direct inhibitory effect on spinal cord dorsal horn neurons and extend the results of previous studies which indicate low frequency EA is mediated by opiate sensitive circuitry, while high frequency EA is predominantly mediated by non-opioid neurotransmitters.

Nitric oxide mediates fos expression in the spinal cord induced by mechanical noxious stimulation

Immunocytochemical localization of Fos protein was used to analyze the involvement of nitric oxide (NO) in the expression of Fos in the spinal cord, induced by mechanical noxious stimulation (NS). Mechanical NS was applied to the left hindpaw 30 minutes after intrathecal administration of the NO synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) and the resulting Fos expression in the spinal cord dorsal horn was compared with that obtained in rats exposed only to the mechanical NS. Pretreatment with L-NAME but not its stereoisomer N omega-nitro-D-arginine methyl ester (D-NAME), produced a dose-dependent suppression of Fos expression induced by mechanical noxious stimulation. These results indicate that NO modulates the expression of Fos in the dorsal horn induced by mechanical noxious stimulation and further support the hypothesis that NO is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus.

Visceral antinociception produced by bee venom stimulation of the Zhongwan acupuncture point in mice: role of α2 adrenoceptors

The goal of the present study was to determine whether bee venom (BV) injection into the Zhongwan acupoint (CV12), compared to injection into a non-acupoint, produced antinociception in an acetic acid-induced visceral pain model. This was accomplished by injecting BV subcutaneously into the Zhongwan acupoint or into a non-acupoint 30 min before intraperitoneal injection of acetic acid in ICR mice. BV injection into the acupoint produced a dose dependent suppression of acetic acid-induced abdominal stretches and of acetic acid-induced Fos expression in the spinal cord and the nucleus tractus solitarii. In contrast BV injection into the non-acupoint only produced antinociception at the highest dose of BV tested. Naloxone pretreatment did not alter the antinociceptive effect of BV acupoint injection on the abdominal stretch reflex. On the other hand, pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine completely blocked the antinociceptive effect of BV acupoint injection. These results imply that BV acupoint stimulation can produce visceral antinociception that is associated with activation of alpha 2-adrenoceptors, but not with naloxone-sensitive opioid receptors.

Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 µg/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.

The anti-inflammatory effect of bee venom stimulation in a mouse air pouch model is mediated by adrenal medullary activity.

Cutaneous electrical or chemical stimulation can produce an anti‐inflammatory effect, which is dependent on adrenal medullary‐sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti‐inflammatory effect that is neurally mediated. In the present study, we examined whether this anti‐inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)‐α concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF‐α elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan‐induced air‐pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti‐inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV‐induced inhibition of leucocyte migration. By contrast, pretreatment with the β‐adrenergic antagonist propranolol reversed the BV‐induced inhibitory effect on leucocyte migration. These results suggest that the anti‐inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.

Spinal neuronal NOS activation mediates sigma-1 receptor-induced mechanical and thermal hypersensitivity in mice: involvement of PKC-dependent GluN1 phosphorylation

BACKGROUND AND PURPOSE We recently demonstrated that activation of the spinal sigma‐1 receptor induces mechanical and thermal hypersensitivity via calcium‐dependent second messenger cascades and phosphorylation of the spinal NMDA receptor GluN1 subunit (pGluN1). Here we examined the role of NO in this process, as it plays a critical role in PKC‐mediated calcium signalling and the potentiation of NMDA receptor function.