Days Postimplantation Research Articles

Device implant based on poly (lactic acid) with vitamin E for vaccine delivery system in Tilapia: Study for biocompatibility and biodegradation

The use of Poly (lactic acid) (PLA) as a slow-release vehicle for vaccines has attracted the attention of researchers, since its insertion improves the uptake of them, and reduces side effects or by stimulating recruited defense cells, assisting immunity without the need for booster vaccine doses. Seeking to develop new strategies for the administration of drugs and vaccines in aquaculture, we evaluated the biocompatibility and biodegradation of polymeric PLA devices and PLA plus vitamin E devices, implanted through subcutaneous (SC) and intraperitoneal (IP) routes in Nile tilapia. To carry out this study, 84 male tilapia (initial 243.82 ± 56.74 g; final 400.71 ± 100.54 g) were randomly distributed in 3 tanks (n = 28 fish per treatment/tank). The devices were prepared in two formulations: neat PLA (containing 100% PLA) and PLAVE (PLA plus vitamin E) implanted using a commercial AnimalTag® applicator, and non-implanted fish (control). Fish were sampled 15, 30, 60, and 120 days post-implantation (DPI). Blood analysis was used to access blood cells and blood smear for differential leucocytes count. Serum biochemistry to evaluated changes in serum proteins and glycemia. Histopathological investigation using hematoxylin-eosin (H&E) was used to assess polymer-tissue interaction. Histochemistry and immunohistochemistry was used to detection immune cells and phagocytes in capsule, and analyses of melanomacrophage centers (MMCs) to morphometric evaluation and percentage amount of melanin, hemosiderin and lipofucsin pigments. Histopathological study revealed an increase of capsular formation and inflammatory cell infiltration in PLAVE-implanted tilapia through SC route (15 DPI). Tilapia implanted with PLAVE and PLA (SC) presented mast cells and eosinophilic granular cells during 15, 30, and 60 DPI, with a decrease in these cells in the fibrous capsule around the polymer at 120 DPI. PLAVE implanted tilapia SC at 60 DPI showed significantly phagocytosis points than other groups. Phagocytic cells (F4/80+) were observed near to biopolymers in phagocytosis sites. Lipofuscin at 120 DPI in spleen melanomacrophage centers were significantly high in PLAVE implanted tilapias when compared to fish with PLA implants and control. The serum biochemical study of tilapia did not reveal changes in cytotoxicity and liver function in implanted fish. The absence of side effects in hematological and biochemical findings, including the absence of mortality after device implantation, proves its clinical safety. PLA implants in tilapia have demonstrated biocompatibility, biodegradation, clinical safety, and excellent evolution of foreign body inflammatory responses.

Clinical safety of zafirlukast treatment during a foreign body inflammatory reaction in nile tilapias, oreochromis niloticus

Seeking to understand the defense mechanisms of tilapia to assist in the sanitary management of fish farms, this study aimed to evaluate the clinical safety of Nile tilapia (Oreochromis niloticus) submitted to oral treatment with 500μg of zafirlukast/Kg of body weight during chronic inflammation, through the implantation of round glass coverslips in the subcutaneous tissue. For this study, 63 male tilapiawere randomly distributed in 9 aquariums to compose the following treatments: Control submitted to inflammatory stimulus without treatment; CT – Conventional treatment; PT – Prolonged treatment. Seven animals were evaluated per treatment in three periods: 2, 4 and 8 days post-implantation (DPI).Tilapia during foreign body reaction showed a decrease in erythrocyte counts and hematocrit values, as well as macrocytic changes, these effects were mitigated by treatment with zafirlukast. Control tilapia showed a gradual increase over time in serum values of total protein, cholesterol, and triglycerides. Serum values of ALT, AST and alkaline phosphatase of control tilapia showed an increase in the late phase of the chronic inflammatory process (8 DPI). Therefore, it was possible to conclude that tilapia treated with 500µg of zafirlukast showed attenuation of hematological changes resulting from fluid-electrolyte imbalance during a foreign body inflammation, as well as ameliorated serum biochemical disorders, demonstrating the clinical safety of oral treatment with this cys-leukotriene blocker.

Survival of heterotopic heart xenografts from Helisoma duryi, Planorbula armigera, and Planorbarius corneus in Biomphalaria glabrata (Pulmonata, Basommatophora, Planorbidae): evidence for phylogenetic relatedness?

Abstract. Heart xenografts from the pulmonate snail Helisoma trivolvis survive in Biomphalaria glabrata, whereas xenografts from most other pulmonate snail genera are rejected within 3 to 15 days. To test whether xenografts from snails closely related to H. trivolvis were also accepted, specimens of an albino strain of B. glabrata were implanted with hearts from H. duryi, Planorbula armigera, and Planorbarius corneus. The fate of implants was monitored for 180 days by measuring heartbeat and by histological analysis. All 3 types of implants survived beyond the 3 to 15 days required for the complete destruction of incompatible xenografts, suggesting a degree of physiological and immunological compatibility between B. glabrata and these donors. Among the 3 types of xenografts, all from H. duryi survived for the entire 180 days. Fewer grafts from P. armigera and P. corneus survived for prolonged periods, although some still were beating at 60 and 180 days post implantation (DPI) respectively, suggesting that a range of histocompatibility, and perhaps phylogenetic relatedness, with B. glabrata occurs within this group. Paradoxically, the initial hemocytic response by the recipient was strongest against grafts from H. duryi, the most compatible donor, while responses to grafts from P. armigera and P. corneus were mild or did not occur during the first 7 DPI. We used 2 albino strains of B. glabrata in these studies, and the data suggest slight differences in recipient strain compatibility.

Short-term immunoisolation of incompatible xenografts in a snail, Biomphalaria glabrata

Hearts of the snails Physa virgata and Biomphalaria glabrata were implanted into the hemocoel of B. glabrata. Implants either were microencapsulated in 2.5% agarose or were unencapsulated. Unencapsulated xenografts from P. virgata underwent complete necrosis within 3 days post-implantation (DPI), whereas allografts were still alive at 7 DPI. In the case of microencapsulated implants, both allografts and xenografts were alive at 3 DPI, showing 4.8 and 14.9% pyknosis among myofiber nuclei, respectively. These results suggest that direct cytotoxicity by hemocytes, rather than toxic plasma factors or donor–recipient physiological incompatibility, is responsible for rapid xenograft death. However, both types of microencapsulated grafts were necrotic at 7 DPI. This necrosis may have resulted from oxygen deprivation brought about both by the agarose and by the heterotopic implantation site, inasmuch as microencapsulated allografts and xenografts cultured in saline for 7 days showed only 1.98 and 30% pyknosis, respectively.

Survival of Heterotopic Headfoot Transplants in Biomphalaria glabrata (Mollusca: Pulmonata)

The entire headfoot (HF) of small snails, Biomphalaria glabrata, was implanted into the hemocoel of allogeneic adults and examined histologically at 30 and 60 days postimplantation (DPI). Although displaying varying degrees of abnormal morphology, the HF allografts continued feeding motions with the buccal mass and showed muscular contractions of the foot muscle. The transected esophagus fused shut and became distended with amorphous material, possibly ingested and/or secreted substances. The buccal mass grew, and pieces of the exfoliated radular ribbon were found in the esophageal sac. Although most major structures of the HF appeared quite normal at the cell and tissue level, the tentacles and epithelium covering the dorsal and lateral surfaces of the HF apparently were lost when in direct contact with the recipient's hemolymph, but persisted if folded or invaginated below the implant surface. No host hemocytic response was observed at either 30 or 60 DPI. These results demonstrate not only considerable physiological plasticity by the implant but also immunological tolerance to histologically complex allografts by the recipient, and support the hypothesis that allorecognition is poorly developed in molluscs. Additional key words: allografts, histocompatibility, snails, tissue transplantation We recently reported survival, for at least 60 days, of several types of allografts in the pulmonate snail Biomphalaria glabrata (SAY), including digestive gland, kidney, mantle, albumin gland, brain, ovotestis, heart, and amebocyte-producing organ (Sullivan et al. 1998). Although pathological changes were observed in implants, these were hypothesized to result from wound healing reactions and degenerative changes (e.g., due to disuse atrophy, accumulation of secretory products, and the physiological unsuitability of the heterotopic location), rather than from an allograft response by the recipient. In fact, little convincing evidence was found for true allograft rejection in molluscs, and previous reports to the contrary may have been due to experimental technique (Sullivan et al. 1998). In this study we examined the fate of larger and histologically more complex transplants. Specifically, we were interested in determining whether the entire headfoot (HF) of small snails would survive if transplanted into the hemocoel of B. glabrata. If such transplants remained viable, then this method could be used to investigate the longevity of sporocysts of Schistoa Author for correspondence. E-mail: sullivan @universe.uiwtx.edu soma mansoni in resistant snails, i.e., by transplanting the HF from schistosome-resistant snails into susceptible recipients, thus allowing viable sporocysts in the implant to complete development. Because of the numerous tissue types, secretions (e.g., mucus, saliva), and relatively large bulk of the HF, we anticipated that such transplants might be problematic. However, the entire implant, as well as its component tissues, survived remarkably well throughout the observation period, with no evidence of rejection by the recipient.

Schistosoma mansoni Infection Inhibits Maturation of Ovotestis Allografts in Biomphalaria glabrata (Mollusca: Pulmonata)

The posterior tip of juvenile snails, consisting of immature ovotestis, vesicular connective tissue, and digestive gland acini, all surrounded by body wall, was implanted into the hemocoel of allogeneic adult Biomphalaria glabrata. Recipient snails were either uninfected or had been infected 15 days previously with Schistosoma mansoni. Graft recipients were dissected at 30 or 60 days postimplantation (DPI), and the implants, along with preimplantation controls, were examined histologically. The gonads in preimplantation controls consisted of a partially coiled collecting canal partitioned dorsally by connective tissue septa and contained mainly spermatogonia-like cells and small oocytes. In uninfected recipients, implanted gonads underwent rapid development, i.e., had formed mature acini and were carrying out vigorous gametogenesis, both spermatogenesis and oogenesis, by 30 DPI. At 60 DPI, numbers of spermatozoa had increased substantially in most implants. In infected recipients, relatively little development or gametogenesis occurred at either 30 or 60 DPI, and oogenesis was more strongly inhibited than spermatogenesis. Except for 1 implanted gonad (out of 25) that had undergone necrosis at 60 DPI and a second otherwise healthy 60-day implant partially encapsulated at its cut surface (both in uninfected recipients), no significant hemocytic response occurred against the allografts.

Comparison of Several Types of Allografts inBiomphalaria glabrata(Mollusca: Pulmonata)

Interpretation of prior studies on molluscan allografts is complicated by the variety of experimental methods used and recurring reports of acute rejection of digestive gland implants. In this study, allografts of adult digestive gland, saccular kidney, mantle, albumin gland, brain, ovotestis, heart, or amoebocyte-producing organ (APO) were implanted into the hemocoel ofBiomphalaria glabratasnails, and their histological condition at 60 days post implantation (DPI) was compared with that of preimplantation tissues. Also, digestive gland tissue, still surrounded by body wall, was implanted from immature donors and examined at 120 DPI. All 60-day allografts maintained some degree of normal cell and tissue structure, and most displayed additional evidence of viability, i.e., contraction (heart), hyperplasia (APO), gametogenesis (ovotestis), secretion (albumin gland, digestive gland, and kidney), shell deposition (mantle), or neuroma formation (brain). Recipient hemocytic reaction was minimal or absent, except that in three of six ovotestis implants and five of seven digestive gland implants, variable numbers of acini were undergoing phagocytosis by hemocytes or had been reduced to granulomas. Among seven implants examined at 120 DPI, digestive gland acini were found in five (with one instance of hemocytic encapsulation). Thus, in this study, acute rejection of allografts did not occur. Although the observed hemocytic reactions could be viewed as evidence of chronic graft rejection, the inconsistent and protracted nature of these responses suggests instead that they represent resorption of degenerating tissue damaged by nonimmunological effects, e.g., mechanical trauma, heterotopic location, or autolysis.

Individual C6 glioma cells migrate in adult rat brain after neural homografting

Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and grafted as a cell suspension (106 cells in 5.0 μl) into freshly made cortical implantation pockets in adult host rats. Animals were killed 1–21 days post-implantation (DPI). The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 μm. Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes. Cells positive for both PHAL and GFAP were graft-derived C6 cells. By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma. This mass increased in size over the next 14 days. The invading tumor mass contained double labeled cells at all time periods examined. In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells). Individual graft-derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.

C6 glioma cell invasion and migration of rat brain after neural homografting

C6 tumor cells (10(6] were grafted as suspensions into freshly made implantation pockets in rat host cerebral cortex. Specimens were prepared for transmission and scanning electron microscopy 1 to 7 days postimplantation (DPI). By 3 DPI vacuolated C6 cells had migrated on or invaded the host brain. C6 cells were observed on the glia limitans on the surface of the brain, in the corpus callosum, subependymal space, and perivascular space and had invaded the cortex under the implantation pocket. In addition to the tumor mass that was observed under the implantation pocket, by 7 DPI individual C6 cells had migrated into the corpus callosum and internal capsule. Migrated C6 cells were observed in a perineuronal position in the hippocampus and other gray matter structures inferior to the corpus callosum. Micropockets were found around each C6 cell and the processes of these cells had replaced host parenchyma. The preferred routes of migration were on basal lamina and parallel and intersecting nerve fiber bundles. Invasion occurred through gray and white matter. The movement of homografted C6 cells in the brain suggests that these cells actively migrate as individual cells in addition to invading as a mass.

Rapid migration of grafted cortical astrocytes from suspension grafts placed in host thoracic spinal cord

The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and homografted as a cell suspension into host thoracic spinal cord. Animals were sacrificed at 7, 14, 21 and 30 days postimplantation (DPI). Paraffin sections of cervical, thoracic and lumbar spinal cord were double-labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes, and PHAL, utilized as a marker for graft-derived cells. PHAL-GFAP positive cells were found throughout the thoracic spinal cord at all time periods, indicating that grafted astrocytes migrated along all 3 axes of the host spinal cord (rostral-caudal, dorsal-ventral and left-right). At 30 DPI, graft-derived astrocytes were found in host cervical and lumbar spinal cord. There appeared to be a minimal delay in the onset of migration.

Fetal cortical astrocytes migrate from cortical homografts throughout the host brain and over the glia limitans.

The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then homografted into freshly made implantation pockets in the host cerebral cortex. Animals were sacrificed at 30 and 60 days postimplantation (DPI). Paraffin sections were double labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes, and PHAL, utilized as a marker for transplant-derived cells. Transplant-derived astrocytes were found on the glia limitans along the entire circumference of the brain, in the hippocampal commissure, corpus callosum, internal capsule, entopeduncular nucleus, habenular commissure, brachium of the superior colliculus, optic tract, optic chiasm, and sensory root of the trigeminal nerve. Transplanted astrocytes entered the spaces of Virchow-Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes.

Ependyma formation in adult rat spinal cord after transplantation of fetal cerebral cortex homografts.

The ependymal cells in rat host spinal cord were studied after transplantation of fetal cerebral cortex homografts. Adult Sprague-Dawley male rats had pieces of E14 fetal rat cortex pressure-injected into the spinal cord at T6. The tissue was prepared for light and electron microscopy and studied for over 2 months postimplantation. At 7 and 14 days postimplantation (DPI), there were dividing ventricular-ependymal cells lining cysts in the host spinal cord. After 21 days, cell division was no longer observed in these cells, and only mature ependymal cells lined these cysts. Mature ependymal cells were either: columnar, pseudocolumnar, cuboidal, squamous, or pseudomorphic; had cilia and villi on only one surface of the cell and shared tight junctions when contiguous. These data show that ependymal cells were mature cellular components of adult spinal cord after transplantation of fetal CNS grafts. In addition, ventricular-ependymal cells continued to divide within the parenchyma of the graft, indicating continued growth of transplanted fetal cerebral cortex in host spinal cord.

Initial growth of transplanted E11 fetal cortex and spinal cord in adult rat spinal cord.

Fetal CNS (cortex and spinal cord) transplants survive and grow in the spinal cord of adult rats. The present experiments study the initial stages (10 days) to determine the developmental pattern of early (E11) fetal cortex and spinal cord transplants implanted into the T6 spinal cord of adult rats (n = 50). Controls were age-matched fetuses or pups from the donor females and surgical and vehicle controls (n = 20). E11 fetal cortex (0.5 X 1.0 mm pieces of neuroepithelium) or spinal cord (1.0 mm) were dissected from caesarean delivered fetuses from time pregnant Sprague-Dawley rats. The fetal tissue was pressure-injected between the dorsal horn and dorsal column of the host. Hosts were prepared for light and electron microscopy 1, 3, 7 and 10 days postimplantation (DPI). At 1 DPI, ovoid, ventriculated multiple neuroepithelia formed in an implantation-induced cyst in the host spinal cord. The inner layer of cells lining the ventricle of the ovoid neuroepithelia was actively dividing. The nuclei of the cells differentiated as they reached the surface of the neuroepithelium. This pattern was repeated at 3-10 DPI. At 7-10 DPI, differentiated neurons and neuroglia surrounded actively dividing neuroepithelia. These cells comprised the bulk of the transplant in the cyst. Transplanted mesenchymal cells formed pockets of cartilage in the implanted spinal cord. Although transplanted CNS consisted of only neuroepithelial cells, they differentiated into neurons that were typical for cortex or spinal cord. These data indicate that the transplanted E11 fetal tissue formed neuroepithelia which established the pattern of growth for these transplants.

Transplantation of fetal rat cortex into regenerating nerve to the biceps femoris of adult rat

Morphological aspects of the implantation of 11-day-old fetal (E11) rat cortex into regenerating peripheral nerves of adult rats were analyzed during the first 3 weeks after transplantation. On day 11 of gestation, donor fetuses were removed from one uterine horn of pregnant Sprague-Dawley rats and pieces of cortex (1.0 × 0.5 mm) were dissected from them for transplantation. Fetuses and normally delivered pups from the other uterine horn were used as age-matched controls. In 45 adult male Sprague-Dawley rat hosts (200 to 300 g), the epineurium of the nerve to the biceps femoris muscle was crushed and incised, the perineurium minced, and E11 donor cortex was injected into the nerve. At 1 to 7 days postimplantation (DPI), undifferentiated neuroepithelial cells were dispersed or in oval neuroepithelial-like structures throughout the regenerating nerve. At 7 to 14 DPI, neurons and neuroglia were differentiating and interspersed among fascicles of regenerating host axons. At 14 to 21 DPI, aggregates of differentiated neurons surrounded by neuropil containing a rich synaptic bed were interspersed with regenerated host peripheral axons. Fetal cortical implants survived, increased in size, and differentiated throughout the postoperative period, matching their timed controls.

Structure and function of fetal cortex implanted into degenerating peripheral nerve of adult rat

The growth, differentiation and function of E11 fetal cortex was studied over 4 months after implantation into the degenerating nerve to the biceps femoris muscle of 70 rats. At 7 days postimplantation (DPI) the implant formed a neuroepithelium which contained mainly undifferentiated cells within a reconstituted perineurium. At 14–21 DPI the neurons contained differentiated, mature nuclei, poorly developed Nissl bodies and normal cytoplasmic organelles. Neuroglia were also observed with typical nuclei and a dense cytoplasmic ground. At 30 days the implants reached their greatest degree of differentiation. Neurons had mature nuclei and cytoplasmic organelles. There were few axosomatic synapses or neuronal cell processes. Neuroglia were prominent with many cell processes. The neuropil contained myelinated and unmyelinated nerve fibers, dendrites and many axodendritic synapses. At 40–50 DPI the neurons and neuronal cell processes were degenerating. At 60–120 DPI the implant contained only neuroglia. Stimulation of only the implant resulted in biceps femoris muscle contraction in 5 animals at 30 DPI and one animal at 40, 45 and 50 days. Injection of 10 μl of WGA-HRP into the biceps femoris muscle resulted in at least 20 neurons in 3 30-day implants that contained HRP reaction product. These data show that fetal cortical neurons can assume functions different from normal, functionally reinnervate adult muscle and can differentiate and form an ectopic nervous system for at least 30-days after implantation into a degenerating adult host peripheral nerve.

Viability, growth, and maturation of fetal brain and spinal cord in the sciatic nerve of adult rat.

The feasibility for growth, maturation, and differentiation of fetal nervous system implanted into adult mammalian peripheral nervous system was studied. Thirty-five adult rats had the epineurium of the sciatic nerve crushed, perineurium minced, and fetal rat cortex or spinal cord implanted. Rats were utilized 7, 14, and 21 days, and 1, 2, 3, and 4 months later. A 1-mm cube of cortex or a 1-mm segment of spinal cord of 11-, 12-, or 15-day gestation fetuses was placed into the epineurium. Age-matched controls (7 DPI (days postimplantation) control for E15 implant was a 1-day pup, 21 days' gestation) were utilized for comparison (two per time group). Five animals had sciatic crush and perineurial mince only, and the gait and toe-spreading response were observed over 4 months. All implanted animals walked normally at 30 days. All implants were successful and survived the duration of the experiment. Cortical implants produced prominent bulges in the epineurium (21 days-4 months). Maximal neuronal and neurolgial cell division was observed at 7-21 days, decreased at 30 days, and stopped by 60 days. Most implanted cortical neurons had mature nuclei and immature dendritic patterns (apolar), and rarely had mature dendritic patterns. Neuroglia were abundant. The younger the cortical implant, the larger the cell mass produced (E11 greater than E15). The spinal cord implants survived, were viable, contained mainly neuroglia, and grew minimally. Host Schwann cells and nerve fibers were found in and around the implants. These data show that adult peripheral nervous system can act as an environment for growth and viability of fetal CNS implants.