Efficient extraction of phycobiliproteins from dry biomass of Spirulina platensis using sodium chloride as extraction enhancer.

An efficient strategy for phycobiliprotein extraction from Spirulina platensis dry biomass has been developed by using NaCl as an enhancer. Different sodium ion and chloride ion salts were screened, and NaCl was selected as the most appropriate solvent for phycobiliprotein extraction. The extraction parameters with NaCl were optimized using response surface methodology. Under optimal operating conditions, a phycobiliprotein extraction rate of 74.8 % and a phycocyanin extraction yield of 102.4 mg/g with a purity of 74.0 % were achieved. Adding NaCl resulted in smaller fragments and destroyed the cell integrity of S. platensis, facilitating phycobiliprotein exudation. The secondary structure and antioxidant activity of phycobiliproteins were not affected by NaCl extraction. The stability of the phycobiliproteins was improved by adding NaCl. This study provides a potential method for phycobiliprotein extraction with high efficiency and good quality using an inexpensive extraction enhancer.

Direct Inhibition of Microglia Activation by Pretreatment With Botulinum Neurotoxin A for the Prevention of Neuropathic Pain.

Peripheral injection of botulinum neurotoxin A (BoNT/A) has been demonstrated to have a long-term analgesic effect in treating neuropathic pain. Around peripheral nerves, BoNT/A is taken up by primary afferent neurons and inhibits neuropeptide release. Moreover, BoNT/A could also be retrogradely transported to the spinal cord. Recent studies have suggested that BoNT/A could attenuates neuropathic pain by inhibiting the activation of spinal glial cells. However, it remains unclear whether BoNT/A directly interacts with these glial cells or via their interaction with neurons. Our aim here is to determine the direct effect of BoNT/A on primary microglia and astrocytes. We show that BoNT/A pretreatment significantly inhibits lipopolysaccharide (LPS) -induced activation and pro-inflammatory cytokine release in primary microglia (1 U/mL BoNT/A in medium), while it has no effect on the activation of astrocytes (2 U/mL BoNT/A in medium). Moreover, a single intrathecal pre-administration of a low dose of BoNT/A (1 U/kg) significantly prohibited the partial sciatic nerve ligation (PSNL)- induced upregulation of pro-inflammatory cytokines in both the spinal cord dorsal horn and dorsal root ganglions (DRGs), which in turn prevented the PSNL-induced mechanical allodynia and thermal hyperalgesia. In conclusion, our results indicate that BoNT/A pretreatment prevents PSNL-induced neuropathic pain by direct inhibition of spinal microglia activation.

Calpain-mediated down-regulation of myelin-associated glycoprotein in lysophosphatidic acid-induced neuropathic pain.

Lysophosphatidic acid receptor (LPA(1)) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA(1) receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA(1) receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.

Evidence for lysophosphatidic acid 1 receptor signaling in the early phase of neuropathic pain mechanisms in experiments using Ki-16425, a lysophosphatidic acid 1 receptor antagonist.

Lysophosphatidic acid is a bioactive lipid mediator with neuronal activities. We previously reported a crucial role for lysophosphatidic acid 1 receptor-mediated signaling in neuropathic pain mechanisms. Intrathecal administration of lysophosphatidic acid (1 nmol) induced abnormal pain behaviors, such as thermal hyperalgesia, mechanical allodynia, A-fiber hypersensitization, and C-fiber hyposensitization, all of which were also observed in partial sciatic nerve injury-induced neuropathic pain. Ki-16425 (30 mg/kg, i.p.), a lysophosphatidic acid 1 receptor antagonist, completely blocked lysophosphatidic acid-induced neuropathic pain-like behaviors, when administered 30 min but not 90 min before lysophosphatidic acid injection, suggesting that Ki-16425 is a short-lived inhibitor. The blockade of nerve injury-induced neuropathic pain by Ki-16425 was maximum as late as 3 h after the injury but not after this critical period. The administration of Ki-16425 at 3 h but not at 6 h after injury also blocked neurochemical changes, including up-regulation of voltage-gated calcium channel alpha(2)delta-1 subunit expression in dorsal root ganglion and reduction of substance P expression in the spinal dorsal horn. All of these results using Ki-16425 suggest that lysophosphatidic acid 1 receptor-mediated signaling which underlies the development of neuropathic pain works at an early stage of the critical period after nerve injury.

Involvement of LPA1 receptor signaling in the reorganization of spinal input through Abeta-fibers in mice with partial sciatic nerve injury.

Lysophosphatidic acid receptor subtype LPA(1) is crucial for the initiation of neuropathic pain and underlying changes, such as up-regulation of Ca2+ channel alpha2delta-1 subunit in dorsal root ganglia (DRG), up-regulation of PKCgamma in the spinal dorsal horn, and demyelination of dorsal root fibers. In the present study, we further examined the involvement of LPA(1) signaling in the reorganization of Abeta-fiber-mediated spinal transmission, which is presumed to underlie neuropathic allodynia. Following nerve injury, the phosphorylation of extracellular-signal regulated kinase (pERK) by Abeta-fiber stimulation was observed in the superficial layer of spinal dorsal horn, where nociceptive C- or Adelta-fibers are innervated, but not in sham-operated wild-type mice. However, the pERK signals were largely abolished in LPA(1) receptor knock-out (Lpar1-/-) mice, further supported by quantitative analyses of pERK-positive cells. These results suggest that LPA(1) receptor-mediated signaling mechanisms also participate in functional cross-talk between Abeta- and C- or Adelta-fibers.

Pharmacological switch in Abeta-fiber stimulation-induced spinal transmission in mice with partial sciatic nerve injury.

BACKGROUND: We have previously demonstrated that different spinal transmissions are involved in the nociceptive behavior caused by electrical stimulation of Abeta-, Adelta- or C-fibers using a Neurometer in naïve mice. In this study, we attempted to pharmacologically characterize the alteration in spinal transmission induced by partial sciatic nerve injury in terms of nociceptive behavior and phosphorylation of extracellular signal-regulated kinase (pERK) in the spinal dorsal horn. RESULTS: Abeta-fiber responses (2000-Hz), which were selectively blocked by the AMPA/kainate antagonist CNQX in naïve mice, were hypersensitized but blocked by the NMDA receptor antagonists MK-801 and AP-5 in injured mice in an electrical stimulation-induced paw withdrawal (EPW) test. Although Adelta-fiber responses (250-Hz) were also hypersensitized by nerve injury, there was no change in the pharmacological characteristics of Adelta-fiber responses through NMDA receptors. On the contrary, C-fiber responses (5-Hz) were hyposensitized by nerve injury. Moreover, Adelta- and C-, but not Abeta-fiber stimulations significantly increased the number of pERK-positive neurons in the superficial spinal dorsal horns of naïve mice, and corresponding antagonists used in the EPW test inhibited this increase. In mice with nerve injury, Abeta- as well as Adelta-fiber stimulations significantly increased the number of pERK-positive neurons in the superficial spinal dorsal horn, whereas C-fiber stimulation decreased this number. The nerve injury-specific pERK increase induced by Abeta-stimulation was inhibited by MK-801 and AP-5, but not by CNQX. However, Abeta- and Adelta-stimulations did not affect the number or size of pERK-positive neurons in the dorsal root ganglion, whereas C-fiber-stimulation selectively decreased the number of pERK-positive neurons. CONCLUSION: These results suggest that Abeta-fiber perception is newly transmitted to spinal neurons, which originally receive only Adelta- and C-fiber-mediated pain transmission, through NMDA receptor-mediated mechanisms, in animals with nerve injury. This pharmacological switch in Abeta-fiber spinal transmission could be a mechanism underlying neuropathic allodynia.

Evidence for the tonic inhibition of spinal pain by nicotinic cholinergic transmission through primary afferents.

BACKGROUND: We have proposed that nerve injury-specific loss of spinal tonic cholinergic inhibition may play a role in the analgesic effects of nicotinic acetylcholine receptor (nAChR) agonists on neuropathic pain. However, the tonic cholinergic inhibition of pain remains to be well characterized. RESULTS: Here, we show that choline acetyltransferase (ChAT) signals were localized not only in outer dorsal horn fibers (lamina I-III) and motor neurons in the spinal cord, but also in the vast majority of neurons in the dorsal root ganglion (DRG). When mice were treated with an antisense oligodeoxynucleotide (AS-ODN) against ChAT, which decreased ChAT signals in the dorsal horn and DRG, but not in motor neurons, they showed a significant decrease in nociceptive thresholds in paw pressure and thermal paw withdrawal tests. Furthermore, in a novel electrical stimulation-induced paw withdrawal (EPW) test, the thresholds for stimulation through C-, Adelta- and Abeta-fibers were all decreased by AS-ODN-pretreatments. The administration of nicotine (10 nmol i.t.) induced a recovery of the nociceptive thresholds, decreased by the AS-ODN, in the mechanical, thermal and EPW tests. However, nicotine had no effects in control mice or treated with a mismatch scramble (MS)-ODN in all of these nociception tests. CONCLUSION: These findings suggest that primary afferent cholinergic neurons produce tonic inhibition of spinal pain through nAChR activation, and that intrathecal administration of nicotine rescues the loss of tonic cholinergic inhibition.

Inhibition of paclitaxel-induced A-fiber hypersensitization by gabapentin.

Paclitaxel (Taxol) is a widely used chemotherapeutic agent in the treatment of several tumors. However, its use is often associated with the generation of peripheral neuropathic pain expressed as mechanical allodynia and thermal hyperalgesia. The molecular mechanism behind this debilitating side effect is obscure, and efficient drugs for its prevention are required. We sought to clarify the cellular changes in the involved nociceptor types underlying paclitaxel-induced neuropathic pain and to test for an alleviating effect of gabapentin treatment in a murine model of paclitaxel-induced neuropathic pain. We found that a single treatment with paclitaxel (4 mg/kg i.p.) led to a decrease in both thermal and mechanical nociceptive thresholds as well as a reduction in the thresholds for 250-Hz (A delta-fiber) and 2000 Hz (A beta-fiber) but not 5-Hz (C-fiber) sine wave electrical stimuli-induced paw withdrawal. The paclitaxel-induced neuropathic pain was completely abrogated by gabapentin (30 mg/kg i.p.) treatment. Furthermore, we found that mRNA and protein levels of the voltage-gated calcium channel (alpha 2)delta-1 subunit (Ca(alpha 2)delta-1), one of the putative targets for gabapentin, was up-regulated in dorsal root ganglions (DRGs), as well as increased expression of Ca(alpha 2)delta-1 protein in medium/large-sized DRG neurons by immunohistochemistry, following paclitaxel treatment. This suggests that paclitaxel induces A-fiber-specific hypersensitization, which may contribute to the functional mechanical allodynia and hyperalgesia, and that gabapentin could be a potential therapeutic agent for paclitaxel-induced neuropathic pain.