Shih-Chan Lai

Immunolocalization of Prophenoloxidase in the Process of Wound Healing in the Mosquito Armigeres subalbatus (Diptera: Culicidae)

Abstract Hemolymph coagulation began almost immediately after wounding in mosquito, Armigeres subalbatus, (Coquillett) larvae. Immunocytochemical localization showed that prophenoloxidase (pro-PO) was distributed in the wound site. In the initial wounding, coagulation and wound plug formation occurred with granulocyte migration. The hemocytes lysed and released granular materials around the wound site, prophenoloxidase being mostly localized in granules and cuticle. In the second phase of wound healing, melanin accumulation occurred at the wound site along the margin of the cuticle and rapidly increased in thickness. Immunogold-labeled pro-PO was localized in vacuoles, melanins, and cuticle, with the gold particles labeled intensely on the undarkened cuticle and weakly on the darkened cuticle. It is believed that pro-PO is activated upon wound initiation to produce melanin product and deposited on the cuticle. In the final phase of healing, scab melanization and pro-PO immunogold localization were reduced and accompanied by epithelial cell regeneration. This proenzyme was localized in vesicles and flocculent materials, but was absent in the melanized scab. Our results further indicate that pro-PO was present in granules, cuticles, epithelial cells, vacuoles, and flocculent materials but not in melanized scab and coagulated clot. The pro-PO immunogold particles labeled intensely in the initial wounding but weakly in the final phase. Our observations also suggest that pro-PO is released from granulocytes by cell rupture, synthesized or stored in granulocytes, and then is released into the wound site via the cytoplasmic granules. This study indicates that the pro-PO is involved in numerous physiological roles in the process of wound healing in this mosquito.

Electron Microscopic Observations on Wound-Healing in Larvae of the Mosquito Armigeres subalbatus (Diptera: Culicidae)

Abstract The wound-healing processes in the mosquito Armigeres subalbatus (Coquillett) were observed with electron microscopy. The initial reaction involved wound contraction and aggregation of injured surface tissues, cell debris and movement of granulocytes toward the wound. Granulocytes first aggregated around the surface of the wound and many filamentous filopodia protruded to connect with cytoplasmic strands. These strands were then interconnected to form a network coagulum resulting in wound closure to prevent body fluid loss. Granulocytes lysed on the wound-site and released granular materials around the wound, inducing localized clot formation. These results suggested that wound-healing in this mosquito species involved both humoral and cellular reactions. The latter reaction involved the movement of plasmatocytes to the basement membrane of the epidermis beneath the wound-site and epithelial cells regeneration. Our observations revealed that wound-healing in A. subalbatus involves the wound contraction, formation of a temporary cellular clump, scar formation, basement membrane formation, and reepithelialization. The larvae neither discarded the wound scar nor secreted a new cuticle until the next molting. Based on the ultrastructural observations, it is suggested that the wound-healing reaction in A. subalbatus was probably a typical response employed by other members of the family Culicidae.

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood-CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.

Albendazole-banlangen Co-therapy against Angiostrongylus cantonensis-induced Eosinophilic Meningitis

The proteolytic enzyme system may play a role in the pathogenesis of angiostrongyliasis meningitis. Although the anthelmintic agent albendazole can kill the Angiostrongylus cantonensis larvae that infect the brain, their dead larvae are capable of evoking a severe, inflammatory response in the brain. Administration of non-steroid anti inflammatory drugs have been reported to possibly relieve the symptoms of meningitis. Thus, this study evaluates the use of the traditional Chinese medicine banlangen in combination with albendazole as co-therapy for eosinophilic meningitis in BALB/c mice. Assay indicators for the therapeutic effect were (1) histopathological changes, (2) Worm recovery, (3)eosinophil counts, and (4) matrix metalloproteinase-9 (MMp-9). We found that the albendazole-banlangen co-therapy significantly decreased these factors (P＜0.05). The combined use of albendazole and banlangen to treat parasitic meningitis may be an effective new means of treating this disease.

Efficacy of N-acetylcysteine or GM6001 Therapy against Angiostrongylus cantonensis-induced Eosinophilic Meningitis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of Angiostrongylus cantonensis-induced eosinophilic meningitis or eosinophilic meningoencephalitis. To study the effect of the anti-oxidant N-acetylcysteine (NAC) or MMP inhibitor GM6001 on this disease, we used A. cantonensis to induce eosinophilic meningitis in BALB/c mice and measured its larvicidal effect, counted leukocytes and measured MMP-9 activity in angiostrongyliasis. We found that GM6001 mildly reduced MMP-9 activity and eosinophils, while NAC did not. We also found no significant difference in the in larvicidal effect in mice treated with GM6001 or NAC (P＞0.05). These findings suggest the effect of therapy with either NAC or GM6001 alone was not significant. GM6001 may be used with an anthelminthic drug to treat parasitic meningitis. However, when using NAC to treat this disease, one must consider NACs ability to cross the blood-brain barrier and get to the target site.

Association of Matrix Metalloproteinases with blood-brain Barrier Damage of Angiostrongyliasis

Blood-brain barrier (BBB) breakdown, causing extravasation of leukocytes into subarachnoid space, is regarded as an important pathophysiological event in meningitis. Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of various inflammatory diseases of the central nervous system. Excessive proteolytic activity of MMP-9 can be detrimental, leading to the disruption of the BBB. The present study showed that the activities of MMP-9 in the cerebrospinal fluid (CSF) were significantly increased in mice with eosinophilic meningitis compared with uninfected mice. CSF eosinophilia was significantly correlated with MMP-9 intensity and CSF total protein. In contrast, MMP-2 was presented and remain unchanged in all CSF samples showing that this enzyme was not correlated with CSF eosinophilia and CSF total protein. In addition, MMP-9 enzymatic activity and CSF total protein were significantly increased upon the establishment of infection but subsided due to an inhibition by GM6001, a specific inhibitor for MMPs, These results suggested that MMP-9 proteolytic activity may be associated with BBB disruption in angiostrongyliasis.

Induction of MMP-9 in Mouse Cerebellum with Purkinje Cell Loss Caused by Angiostrongylus Cantonensis

Neurological disorders in angiostrongyliasis are caused by invasion of the central nervous system by developing Angiostrongylus cantonensis larvae. After A. cantonensis infection, the cerebellums of BALB/c strain mice were observed to have a loss of Purkinje cells. Histologically, the Purkinje cells in the infected mice cerebellum were shown to have degenerative atrophy or partial loss. The matrix metalloproteinase-9 (MMP-9) mRNA and activity was induced in the infected mice, and the enzyme was absent in uninfected mice. Western blotting showed that the anti-MMP-9 antibody recognized a single 94 kDa protein from A. cantonensis-infected mice. Immunohistochemistry also showed localization of MMP-9 within the degenerative Purkinje cells. These results suggest MMP-9 in mice infected with A. cantonensis may be related to the loss of Purkinje cells.