Zhao-Liang Xin, Neurosurgery Department of Yiwu Central Hospital, 3^rd Faculty of Medical College, Zhejiang University, Yiwu 322000, ZheJiang Province, China
Song-Lin Ge, 3^rd Faculty of Medical College, Zhejiang University, Hangzhou, 310016, ZheJiang Province, China
Xiao-Kang Wu, Department of Neurosurgery, Affiliated Hospital of Railway University, Shanghai, China
Yan-Jie Jia, Han-Tao Hu, Department of Anatomy, Medical College, Xi'an Jiaotong University, Xi'an 710007, Shaanxi Province, China
Supported by the Natural Science Foundation of Shaanxi Province, No. 98SZ-064
Correspondence to: Zhao-Liang Xin, Yiwu Central Hospital, Yiwu 322000, Zhejiang Province, China.  wxk555@sohu.com
Telephone: +86-579-5209626    Fax: +86-579-5209618
Received: 2004-08-26    Accepted: 2004-12-21

Abstract
Aim: To identify the effect of decreasing the rejection and the death reason of implanted islets encapsuled by biological semi permeable membrane (BSM) in combined with the privileged site of xenotransplantation.

Methods: After the BSM experimented in vitro, the SD rat’s islet-like cell clusters (ICCs) encapsuled by BSM were xenotransplanted into normal dog’s brain. Morphological changes were observed by light and transmission electron microscope. The islets and its apoptosis of implants were identified by insulin-TUNEL double staining of immunoh-istochemistry.

Results: The BSM used in our research had favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts were made up of epithelioid cells and loose connective tissue, heavy infiltration of inflammatory cells was not observed, the implanted B cells were identified 2 mo later and showed a classic presentation of apoptosis.

Conclusion: Using the BSM in combination with the privileged site xenotransplantation have a beneficial effect on the inhibition of the rejection of heterogeneous ICCs implanted, and the death of implanted heterogeneous B cells are associated with apoptosis.

ã 2005 The WJG Press and Elsevier Inc. All rights reserved.

Key words: Islets of Langerhans; Xenotransplantation; Brain; Semi permeable membrane

Xin ZL, Ge SL, Wu XK, Jia YJ, Hu HT. Intracerebral xenotranspl-antation of pancreatic islets encapsulated in a semi permeable membrane. World J Gastroenterol  2005; 11(36): 5714-5717
http://www.wjgnet.com/1007-9327/11/5714.asp

INTRODUCTION
Studies have shown that the porcine ICCs xenotransplanted into rat’s brain could survive for only eighteen days^[1,2], which suggests that the immuno-privileged function of brain cannot ensure the long-time survival of the heterogeneous islets. Immunity-isolation is an effective way to lessen the immunological rejection damage^[3,4]. Better effect would have been derived through the combination of different immunity-isolation technique^[5]. The rat’s pancreatic islet is easy to be separated, the transgenic rats are also developed, and the cell line that can secrete insulin has been established^[6]. Otherwise, the exactl effects of apoptosis in the death of islets xenotransplanted are not well known^[7-9]. In this study, the SD rat’s ICCs encapsuled by a BSM (made by us) were xenotransplanted into a normal dog’s brain to identify the effect of decreasing the rejection and the death reason of implanted islets; this will provide experimental proof for the later clinical application.

MATERIALS AND METHODS
Separation, purification and culture of rats ICCs ^[6]
SD rats, weighing from 150-200 g, were used in this study. Pancreas was removed with the amiculas and vessels removed. They were washed twice in Hank’s, and then minced. The fragments were digested with collagenase V (1 g/L, Sigma) at 37 ℃ for 15-20 min, then were grinded and filtrated on the 80-mesh steel nets to remove connective tissue, at the same time cold Hanks was used to wash the fragments and stop the activity of collagenase. Then the filtrates was filtrated with 400-mesh steel nets to remove the small exocrine cells, red blood cells and white blood cells, the bigger ICCs were kept on the net. The cell clusters on the net were collected and put into the Ficoll fluid (Euro-Collin) whose concentration gradients were 25, 23, 20 and 11 g/L. The mixed liquors were centrifuged at 3 500 r/min at 4 ℃ for 15 min. Most ICCs were gathered at the interface of 23-20 g/L and 20-11 g/L, and then the ICCs were washed thrice. Purity of ICCs acquired by this way was (88.27.6)%. These ICCs were cultured in non-insulin DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 mmol/L nicotinamide, 5 mmol/L hydroxyethyl methylmethane sulfonic acid, 100 000 U/L penicillin and 1 g/L streptomycin. They were maintained at 37 ℃ in a high-humidity incubator with 50 mL/L COs₂ atmosphere. Four hundred to thousand purified ICCs were obtained from every rat. These ICCs were stained by trypan blue and the activity was 90%.

In vitro experiments of BSM
Lymphocytes diffusion  Five semi permeable membrane encysts were filled with 1 mL of fresh blood plasma and put in 200 mL of blood. Then they were maintained at 37 ℃ in a high-humidity incubator with 50 mL/L CO₂ atmosphere and were kept agitated with magnetic force. Fluid in encysts was suctioned respectively 5 h later, then smeared and observed through a light microscope by Wright’s staining.
Glucose diffusion  Eight semi permeable membrane encysts were filled with 1 mL of isotonic sodium chloride, put in 400 mL of isotonic sodium chloride containing 29.1 mmol/L glucose, and kept agitated with magnetic force. The encysts were taken out respectively at 1, 4, 8, 12, 16, 20, 24 and 28 min and washed with isotonic sodium chloride. The fluid in the encysts as obtained and the concentration of glucose was detected.
In vitro culture of ICCs encapsulated by BSM  About twelve thousand separated ICCs were divided into ten parts, five were encapsulated by BSM, five were routinely cultured, the culture mediums used were as before, which were changed every three days. The concentration of insulin in the medium was detected respectively on d 3, 6, and 15 at the moment of changing the medium.

SD rats ICCs encapsulated by BSM transplanted into dog’s brain
ICCs cultured for 72 h were encapsulated by aseptic BSM. The volume of encysts was 1.5 cm×1.5 cm×1.5 cm. Each of them contained 6 000 to 10 000 ICCs.
    The recipients were normal male local hybrid dogs weighing from 7 to 14 kg. The dogs were intravenously anesthetized and hair was shaved. The straight nick was made at frontal-occipital middle line, which was perpendicular to the zygomatic arch. The temporalis muscles was pulled back, the skull was exposed, a hole was burred, the skull window was enlarged to 3 cm×3 cm, and the dura mater was cut open. The pallium was exposed, a nick was made under the cerebral lateral fissure, and between the rectal fissure and rectal lateral fissure, the fistula into the ventricle formed a 2 cm×2 cm×1.5 cm tunnel. The brain tissue was wet compressed by 0.5 g/L orthophosphoric acid dexamethasone for 15 min^[7], then the encysts were transplanted into the tunnel. The dura mater was relocated and compressed by gelatin sponges from outside. Scalp and muscles were then sutured. Dogs could take food when consciousness was regained after anesthesia. Penicillin and streptomycin was used 30 min before the operation and 1-3 d after the operation.
    Transplant dogs were divided into two groups: Control group (n = 2): After making a fistula, the stoma at the ventricle side was covered by gelatin sponges. Then the encysts were cut open to let the contents efflux into the brain tissue and the stoma at cortex side was covered by gelatin sponges. Experimental group (n = 4): Brain tissue was obtained 1 mo and 2 mo after encapsulated ICCs had been transplanted.

Observation Items
To observe morphological changes According to the schedule, 4 g/L paraformaldehyde phosphate buffer was used to perfuse the brain through the ambi-common carotid artery, and then the brain was obtained. The grafts shape, color, location and the changes of surrounding brain tissue was generally observed. Then part of the grafts and surrounding brain tissue were paraffin imbedded, sectioned and stained with HE. Morphological changes of grafts and brain tissue were observed though a light microscope. Some grafts were divided into pieces of 1 mm×1 mm×1 mm. These pieces were fixed by 25 g/L glutaraldehyde and dehydrated, embedded, then ultrathin sectioned. The ultramicro-changes of the grafts were observed by a transmission electron microscope (JEM 2 000).
To identify the islets and its apoptosis of implants An immunohistochemistry staining group and a negative control group were set, normal mouse serum replaced the first antibody in the negative control group. Paraffin imbedding section was used. The first antibody is mouse anti-human insulin used at 1:100 dilution. It was immunohistochemistry stained according to the avidin-biotin compound method (ABC), DAB coloration, and then hematoxylin counterstained to identify B cells in the grafts. The DNA fragmentation of B cells of implants was identified by insulin-TUNEL double staining of immunohistochemistry.

RESULTS
In vitro experiments of BSM
The BSM was not damaged during the whole process of the experiment in vitro. In the lymphocyte-diffusion experiment, it was not observed that lymphocytes and any other blood cells immersed into the semi permeable membrane capsule, but glucose could diffuse into the capsule rapidly. The concentration of glucose in the capsule was 14.3 mmol/L after 8 min, which is 50% of the concentration out of the capsule. After 28 min, the concentration of glucose in the capsule achieved the same level of it in the environment. 
    The concentration of insulin in the medium of experi-mental group on 3^rd, 6^th and 15^th d were (139.0±3.4) mU/L, (78.7±3.9) mU/L and (66.2±2.9) mU/L. That of the control groups are (145.4±4.2) mU/L, (79.4±2.1) mU/L and (48.2±4.1) mU/L. There was not conspicuous variability (P>0.05) between two groups at various times. Insulin secreted by pancreatic islet cells could diffuse rapidly out of the capsule.

ICC xenotransplanted into dog’s brain
The dogs of both groups did not have conspicuous behavior disorder. None of the dogs in the experimental group was infected.
    The appearance of the graft was deep gray and homogenous in experimental group. It was connected with the ventricle at the inner side and adhered with thickening dura mater at the lateral side. It was in contact with the cerebral parenchyma. The boundary was clear. Brain tissue around the graft did not have the obvious appearance of hemorrhage, necrosis and inflammatory abscess. Only residuary semi permeable membrane was observed in the dog’s brain of control group.
    Under light microscopy, the graft was made up of epithelial cells and loose connective tissue, including epithelial cells, fibroblasts, collagen fiber, and small vessels. In the middle of them, there were lymphocytes and other kind of white cells. Epithelial cells mostly stuck on fibroblasts, collagen fiber or small vessels. They were arranged loosely in the connective tissue according to the direction of the collagen fibers. The structure of small vessels was normal. The structure of some parts of the semi permeable membrane was different from it when transplanted, but there was not clump-like infiltration of lymphocytes here. Slight hyperplasia and hydropsia of glial cells were observed in the brain tissue around the graft, but heavy infiltration of inflammatory cells was not observed.
    Under electron microscopy, secreting granulas were observed in the endochylema. The granulas were not of the same size and not identical with the granulas of pancreatic islet’s A, B and C cells. Caryons of some cells were intact, others were contracting, chromosome aggregating, and the typical apoptosis bodies were also found (Figure 1).

Figure 1  Under electron microscopy, typical appearances of apoptosis in transplant cells were found at 1 mo of transplant. (6 000) ↑collagen fiber, ↑WBC, ▲ apoptosis body.

The xenotransplanted cells had disappeared in the control group.
In the immunohistochemistry staining, the cytoplasms of many epithelial cells in the grafts of EG were stained as yellow, which indicated that they were islet B cells (Figure 2). We found that the implanted B cells showed a classic presentation of apoptosis by insulin-TUNEL double staining.

Figure 2  The cytoplasm of some cells in the grafts of experimental group were stained as yellow by the anti-insulin antibody at 1 mo of transplant, which indicated that they were islet B cells (400).

DISCUSSION
Studies have shown that porcine ICCs xenotransplanted into a rat’s brain could survive for only 18 d^[1,2], and fetal porcine ICCs transplanted into cynomolgus monkey’s kidney encyst were completely rejected until the 6^th d^[5,8]. This study showed that there was not infiltration of clump-like polymorphic nucleus cells and lymphocytes, which mark the occurrence of rejection, immunohistochemistry staining showed that pancreatic islet B cells still existed in the grafts after 2 mo; the dogs did not have conspicuous behavior disorder after transplantation. Slight hyperplasia and hydropsia of glial cells were observed in the brain tissue around the graft. These facts verified that using the BSM in combination with the privileged site of xenotransplantation have a beneficial effect on the inhibition of the rejection of heterogeneous ICCs implanted.
    Cells display different morphological features in different environments to adapt themselves to the microenvironment. The culture medium was gradually absorbed after grafting and replaced by loose connective tissue until solidification. The appearance of connective tissue is a kind of compensatory reaction; it gives support and nutrition to islet cells and keeps them stable. So islet cells were arranged loosely in the connective tissue according to the direction of collagen fibers, instead of the in situ compact cell clumps, which are under the pressure of exocrine division.
    It is easier to transplant, when pancreatic islets are capsuled and easier to dislodge, when the rejection is serious during the early period of transplantation. This is very important when a clinical trial is carried out. According to the in vitro experiments and transplant experiments, the semi permeable membrane used in our research has favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity.
    Some studies had cerebrospinal fluid (CSF) which drained from the ventricle to the abdominal cavity, islets was loaded in a chamber connected to the shunt, and CSF could ensure islet survival with sufficient oxygen and nutrients^[10]. We made a tunnel into the ventricle, which connect capsules with CSF immediately. Accordingly, islets were supplied with nutrients in the earlier period, which was beneficial for their survival. However, it is unknown how many islet nutrients acquired this way can be supported. We also found that the implanted B cells showed a classic presentation of apoptosis by insulin-TUNEL double staining and the electron microscopy, the death of implanted heterogeneous B cells is associated with apoptosis. What is the reason of apoptosis and how to delay or block this apoptosis are the subjects to be studied^[11-14].
    Operative procedures injure brain tissue and the integrality of the blood-brain barrier. Wet compressing of dexamethasone on the injured brain can lower the permeability of the blood-brain barrier and decrease non-specific exudation of inflammatory cells^[15].

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