Efficient hepatocyte engraftment in a nonhuman primate model after partial portal vein embolization

BACKGROUND: Hepatocyte transplantation could be an alternative to whole liver transplantation for the treatment of metabolic liver diseases. However, the results of clinical investigations suggest that the number of engrafted hepatocytes was insufficient to correct metabolic disorders. This may partly result from a lack of proliferation of transplanted hepatocytes. In rodents, portal ligation enhances hepatocyte engraftment after transplantation. We investigated the effects of partial portal ligation and embolization on engraftment and proliferation of transplanted hepatocytes in primates. METHODS: Hepatocyte autotransplantation was performed in Macaca monkeys. The left lateral lobe was resected for hepatocyte isolation. The first group of monkeys underwent surgical ligation of the left and right anterior portal branches; in the second group, the same portal territories were obstructed by embolization with biological glue. To evaluate the proportion of cell engraftment hepatocytes were Hoechst-labeled and transplanted via the portal vein. Cell proliferation was measured by BrdU incorporation. RESULTS: Hepatocyte proliferation was induced by both procedures but it was significantly higher after partial portal embolization (23.5% and 11.2% of dividing hepatocytes on days 3 and 7) than after ligation (3% and 0.8%). Hepatocytes engrafted more efficiently after embolization than after ligation. They proliferated and participated to liver regeneration representing 10% of the liver mass on day seven and their number remained constant on day 15. CONCLUSIONS: These data suggest that partial portal embolization of the recipient liver improves engraftment of transplanted hepatocytes in a primate preclinical model providing a new strategy for hepatocyte transplantation.     

Liver repopulation and long-term function of rat small hepatocyte transplantation as an alternative cell source for hepatocyte transplantation.

Hepatocyte transplantation (HT) is an attractive therapeutic modality for liver disease as an alternative for liver organ transplantation. Primary fresh hepatocytes (FHs) are the exclusive cell source that has been used for clinical HT. However, the use of FHs is limited due to a shortage of donor cells. Small hepatocytes (SHs) are hepatic progenitor cells and can be isolated not only from rodents but also from humans. SHs can proliferate in vitro and express liver functions, although conventional hepatocytes lose them within a short period after culture. SH functions in vivo have never been studied. We therefore investigated HT using SHs to evaluate cell engraftment and function compared to HT using FHs. The donor cell number in the SH group was smaller than that in the FH group at HT. The cell engraftment in the SH group was smaller in the liver and larger in the spleen than in the FH group. The cell engraftment in the liver increased after HT; however, that in the spleen decreased after HT in both groups. HT using SHs supported the serum albumin level in the NAR experiment as well as that using FH, and albumin mRNA was detectable in the recipients' tissues at 12 weeks after HT. In conclusion, HT using SHs showed hepatic repopulation similar to that using FHs. This suggests that both SHs and FHs can repopulate the liver as if they were hepatic stem cells. In addition, HT using SHs supported liver functions such as albumin correction at the same level as that using FHs. These observations strongly support the idea that SHs could be an alternative to primary FHs as a novel cell source for future HT.     

Hepatocyte transplantation for total liver repopulation

Hepatocyte transplantation (HT) is an attractive therapeutic alternative to liver transplantation. A number of experiments have shown the feasibility of total liver parenchymal cell replacement by transplanted hepatocytes. In this review, we would like to highlight researches and clinical reports of HT for liver repopulation. Cellular source of clinical HT should be safety. Immortalized cells, hepatic stem cells, and other stem cells have been used for an experimental model for HT. The exact mechanism of the cell engraftment after HT has not been completely understood, although there were some markers to detect and investigate transplanted cells. In order to achieve liver repopulation following HT, a mild hepatic damage may need to facilitate cell engraftment and replace the host liver by transplanted cells. Hormonal factor may use for the same purpose. Despite the results of preclinical studies promising clinical benefits for cell therapy, the clinical experience of HT has been disappointing, except in a few cases. HT may become an alternative for liver transplantation in the future; however, many efforts should made before establishing an effective method for HT and liver replacement therapy.     

Portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient

Hepatocytes transplantation is viewed as a possible alternative or as a bridge therapy to liver transplantation for patients affected by acute or chronic liver disorders. Very few data regarding complications of hepatocytes transplantation is available from the literature. Herein we report for the first time a case of portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient. A patient affected by acute graft dysfunction, not eligible for retransplantation, underwent intraportal infusion of 2 billion viable cryopreserved ABO identical human allogenic hepatocytes over a period of 5 h. Hepatocytes were transplanted at a concentration of 14 million/ml for a total infused volume of 280 ml. Doppler portal vein ultrasound and intraportal pressure were monitored during cell infusion. The procedure was complicated, 8 h after termination, by the development of portal vein thrombosis with liver failure and death of the patient. Autopsy showed occlusive thrombosis of the intrahepatic portal vein branches; cells or large aggregates of epithelial elements (polyclonal CEA positive), suggestive for transplanted hepatocytes, were co-localized inside the thrombus.     

The role of progenitor cells in repair of liver injury and in liver transplantation

There are three levels of cells in the hepatic lineage that respond to injury or carcinogenesis: the mature hepatocyte, the ductular "bipolar" progenitor cell, and a putative periductular stem cell. Hepatocytes are numerous, and respond rapidly to liver cell loss by one or two cell cycles but can only produce other hepatocytes. The ductular progenitor cells are less numerous, may proliferate for more cycles than hepatocytes, and are generally considered "bipolar," i.e., they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential, and may be multipotent. Extrahepatic (bone marrow) origin of the periductular stem cells is supported by recent data showing that hepatocytes may express genetic markers of donor hematopoietic cells after bone marrow transplantation. These different regenerative cells with variations in potential for proliferation and differentiation may provide different sources of cells for liver transplantation: hepatocytes for treatment of acute liver damage, liver progenitor cell lines for liver-directed gene therapy, and bone marrow-derived cells for chronic long-term liver replacement. A limiting factor in the success of liver cell transplantation is the condition of the hepatic microenvironment in which the cells must proliferate and set up housekeeping.     

State of hepatocyte transplantation: a risk or a chance?

Over the past few years, hepatocyte transplantation has been considered as an alternative method for orthotopic liver transplantation for the treatment of various liver diseases. Beside curative approach for genetic metabolic deficiencies (familial hypercholesterolemia, hemophilia, etc.), it could be a useful tool for bridging the waiting period until an appropriate donor organ is obtained. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally or into the spleen settle down in the diseased liver. This enables genetic modification to correct inborn metabolic deficiencies and improves survival in acute liver failure. In 1992, the first clinical transplantation of isolated hepatocytes in 10 patients was performed. In 1998, Fox and coworkers described the successful transplantation of allogeneic liver cells in a child with Crigler-Najjar syndrome. Accomplished studies of Strom et al. resp. Bilir et al. of the same year proved the effectiveness of liver cell transplantation for transient treatment of acute liver failure. Prerequisite of this cell-based therapeutic strategy is a sufficient amount of highly differentiated hepatocytes, hence, a well established in-vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes and to preserve the physiological cell function. This review discusses the different experimental approaches regarding the cultivation of human hepatocytes and also the use of alternative cell sources (like animal hepatocytes, immortalized cells of human origin, progenitor cells from fetal human liver/liver stem cells) for hepatocyte transplantation.     

Improving the techniques for human hepatocyte transplantation: report from a consensus meeting in London

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.     

One liver, three recipients: segment IV from split-liver procedures as a source of hepatocytes for cell transplantation

Hepatocyte transplantation is emerging as a possible treatment for patients with acute liver failure and liver-based metabolic disorders. With the limited availability of donor tissue, it is important to find new sources of liver tissue for isolation of high-quality hepatocytes. Segment IV with or without the caudate lobe was removed during three split-liver procedures. Hepatocytes were isolated from the tissues using a collagenase perfusion technique under strict sterile conditions. The mean number of hepatocytes that were isolated was 5.14 x 10(8) cells with a mean cell viability of 89%. Two of the hepatocyte preparations were used for cell transplantation in a 1-day-old boy with an antenatal diagnosis of a severe urea cycle defect caused by ornithine transcarbamylase deficiency. The six recipients of split-liver grafts demonstrated no complications related to the removal of segment IV. Segment IV with or without the caudate lobe obtained from split-liver procedures is potentially a good source of high-quality hepatocytes for cell transplantation.     

猪急性缺血性肝衰模型的建立和辅助性异位部分肝移植的作用

目的: 探讨急性缺血性肝衰模型的制备、辅助性异位部分肝移植的作用. 方法: 用家猪配对开展辅助性异位部分肝移植.分三组,A组:受体肝脏和肝动脉保持原状,其门静脉缩窄;供肝植入受体右肝下,仅建立门静脉血供,不建立动脉血供.B组:受体肝动脉结扎,其他手术内容与A组相同.C组:受体肝动脉结扎,供肝动脉和门静脉血供均建立,其他手术内容与A组相同.监测各组受体存活情况,肝功能和肝脏血流情况,病理及供肝胆汁分泌情况. 结果: A组、C组受体3 d以上成活率显著高于B组.A组、C组手术前后胆红素无显著改变,B组术后胆红素显著高于术前,术后第二天B组胆红素显著高于A组、C组.C组供肝胆汁分泌和血供良好,肝细胞存活并有活跃的代偿性增生;A组、B组供肝无或仅有少量胆汁分泌,肝细胞大片坏死. 结论: 受体肝动脉结扎、门静脉缩窄足以造成急性肝衰模型;保留受体肝脏动脉血供、减少门静脉血供对受体肝脏功能无严重影响;辅助性异位部分肝移植能取得良好的效果,足以纠正急性肝衰.     

Engraftment measurement in human liver tissue after liver cell transplantation by short tandem repeats analysis

Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/ recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR Profiler Plus PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.     

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.     

Hepatocyte transplantation in the Long Evans Cinnamon rat model of Wilson's disease

Wilson's disease (WD), caused by a mutation in the P-type copper transporting ATPase (Atp7b) gene, results in excessive accumulation of copper in the liver. Long Evans Cinnamon rats (LEC) bear a mutation in the atp7b gene and share clinical characteristics of human WD. To explore hepatocyte transplantation (HT) as therapy for metabolic liver diseases, 8-week-old LEC rats (n = 12) were transplanted by intrasplenic injection of hepatocytes from donor Long Evans (LE) rats. Immunosuppression was maintained with intraperitoneal tacrolimus. The success of HT was monitored at 24 weeks of life. Serum aminotransferases and bilirubin peaked at 14-21 weeks in both HT rats and nontransplanted controls, but at 24 weeks, survival was 97% in LEC-HT versus 63% in controls. All transplanted rats showed restored biliary copper excretion and reduced liver iron concentration associated with increased ceruloplasmin oxidase activity. Liver tissue expressed atp7b mRNA (11.9 +/- 13.6%) indicative of engraftment of normal cells in 7 of 12 HT rats, associated with a reduced liver copper concentration compared to untreated LEC rats. Periportal islets of normal appearing hepatocytes, recognized by atp7b antibody, were observed in transplanted livers while lobular host cells showed persistent pleomorphic changes and inflammatory infiltrates. In conclusion, transplantation of normal hepatocytes prevented fulminant hepatitis, reduces chronic inflammation, and improved 6-month survival in LEC rats. Engraftment of transplanted cells, which express atp7b mRNA, repopulated the recipient liver with normal functional capacity.     

A novel system for transplantation of isolated hepatocytes utilizing HBsAg-producing transgenic donor cells

Hepatocytes from donor transgenic mice that produce an easily assayable circulating marker have been used to develop a novel hepatocyte transplantation system. Isolated G7 HBV transgenic donor hepatocytes secreting HBsAg were transplanted into congeneic or allogeneic mouse recipients. Serum HBsAg was present three days after hepatocyte transplantation in congeneic animals and persisted indefinitely when hepatocytes were transplanted into the spleen. Transplanted hepatocytes within the splenic pulp were identified by morphologic and histochemical analysis. Migration of hepatocytes injected into the spleen to the liver was demonstrated by in situ hybridization using an RNA probe for HBsAg. Transplantation into nonimmunosuppressed allogeneic recipients resulted in disappearance of detectable hepatocytes in the spleen within two weeks. This novel transplantation system should facilitate studies of hepatocyte engraftment and survival, modulation of allograft rejection, and development of hepatocyte-directed gene therapy.     

Liver Repopulation: A New Concept of Hepatocyte Transplantation

Hepatocyte transplantation has been recognized as an alternative strategy for organ transplantation because the supply of donor livers is limited. However, in conventional hepatocyte transplantation, only 1%–10% of the liver replaced with transplanted hepatocytes. Recently a novel concept termed “liver repopulation” has been established, where the whole recipient liver can be replaced by a small number of donor hepatocytes. To induce liver repopulation, growth advantage of the donor hepatocytes against the host liver seems to be required according to the data of previous studies. Additionally, various cell sources, including bone marrow cells and other stem cells, could potentially be used as donor cells for liver repopulation. In this article, we discuss recent progress and future perspectives of this emerging technology.     

Transplantation of hepatocytes in nonhuman primates: a preclinical model for the treatment of hepatic metabolic diseases

BACKGROUND: The transplantation of isolated hepatocytes in large animals, including nonhuman primates, must be evaluated before clinical trials are performed. However, in the absence of large transgenic animals and large-animal (as opposed to small-animal) models of genetic deficiencies, it is difficult to evaluate the fate of transplanted hepatocytes, their localization, survival, and function within the parenchyma of the host liver. In this work, we aimed to develop a technique for delivering hepatocytes to the liver of a nonhuman primate and to evaluate their localization and functionality in the short term. METHODS: A 20% hepatectomy was performed in 34 cynomolgus monkeys (Macaca fascicularis) and hepatocytes were isolated. Hepatocytes were labeled in vitro with a recombinant retrovirus expressing the beta-galactosidase gene and returned to the liver by infusion through a portal catheter left in place. Liver biopsies were performed 4 and 7 d after transplantation. RESULTS: Twenty-four monkeys underwent surgery to define the necessary technical adjustments and to optimize conditions. Six monkeys died. The whole protocol, including the transplantation of genetically marked hepatocytes and procurement of liver biopsies, was performed in the remaining 10 monkeys. In eight monkeys, transplanted hepatocytes expressing the beta-galactosidase gene were widely distributed in the portal tracts, sinusoids, and hepatocyte plates of the host liver 4 and 7 d after transplantation. CONCLUSIONS: We have developed an experimental nonhuman primate model for the evaluation of hepatocyte transplantation. We demonstrated the engraftment and functioning of transplanted hepatocytes in the host liver 4 and 7 d after transplantation.     

Are hematopoietic stem cells involved in hepatocarcinogenesis?

The liver has three cell lineages able to proliferate after a hepatic injury: the mature hepatocyte, the ductular “bipolar” progenitor cell termed “oval cell” and the putative periductular stem cell. Hepatocytes can only produce other hepatocytes whereas ductular progenitor cells are considerate bipolar since they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential and may be multipotent, being this aspect still under investigation. They originate in the bone marrow since their progeny express genetic markers of donor hematopoietic cells after bone marrow transplantation. Since the liver is the hematopoietic organ of the fetus, it is possible that hematopoietic stem cells may reside in the liver of the adult. This assumption is proved by the finding that oval cells express hematopoietic markers like CD34, CD45, CD 109, Thy-1, c-kit, and others, which are also expressed by bone marrow-derived hematopoietic stem cells (BMSCs). Few and discordant studies have evaluated the role of BMSC in hepatocarcinogenesis so far and further studies in vitro and in vivo are warranted in order to definitively clarify such an issue.     

Human hepatocyte isolation and relationship of cell viability to early graft function

Hepatocyte transplantation is emerging as an additional modality of treatment for patients with acute liver failure or liver-based metabolic disorders. The procedure requires isolation of high-quality hepatocytes from unused donor livers. Hepatocytes were isolated from 20 donor livers (11 right lobes, 3 left lateral segments, 6 whole livers) using a collagenase perfusion technique. Cell viability (median 56%, range 13-95%) and yield (median 1.4 x 10(9) cells, range 2.0 x 10(6)-1.8 x 10(10) cells) varied according to the tissue available. Fatty livers rejected for transplantation gave lower cell viability (median 45%, range 25-59%). There was a significant correlation between age of donor (median 21 years, range 7-66 years) and viability of isolated hepatocytes in vitro (r = -0.683, p = 0.001). The 13 segments of livers were from reduced/split grafts used for clinical transplantation in 9 children and 4 adults. There was no significant correlation between in vitro cell viability and clinical parameters including intensive care stay, serum aspartate aminotransferase,and international normalized ratio (in the first 7 days), and allograft rejection or other early posttransplant complications, in patients transplanted with the corresponding tissue.     

Technical aspects of portal vein arterialization for acute liver failure: from rat lab to man

Survival rates of patients with acute liver failure (ALF) without transplantation are poor. However, many of them die awaiting a transplant because of the donor organ shortage. Supporting these patients until an organ becomes available or until their own liver is able to regenerate itself thus avoiding transplantation is a major goal in their multidisciplinary treatment. Animal experimental studies have shown that portal vein arterialization (PVA) enhances the regenerative capacity of hepatocytes by increasing the oxygen supply to the liver after extended hepatectomy or in toxin-induced ALF models. Furthermore, we have reported the application of PVA in patients with ALF. We herein have described the technical aspects of the PVA procedure both in preclinical models and in man.     

Intrasplenic transplantation of allogeneic hepatocytes modified by BCL-2 gene protects rats from acute liver failure

BACKGROUND: Apoptosis of donor hepatocytes may be induced by recipient cytotoxic T lymphocytes (CTLs) during acute rejection, representing a major impediment for these cell transplants. Because the mechanisms of transplanted hepatocyte loss involve Fas-mediated pathways, BCL-2 genetic modification may protect liver cells. In the present study, we further investigated whether BCL-2 transfer into transplanted liver cells rendered them resistant to Fas ligand-induced apoptosis, and protected rats from acute liver failure. MATERIALS AND METHODS: Hepatocytes isolated from Sprague-Dawley rats were infected with an adenovirus vector encoding human BCL-2 gene (AdCMVBCL-2) or a control AdCMVLacZ vector. Forty-eight hours later, cells challenged with recombinant Fas ligand (rhsFasL) were assayed for apoptosis using TUNEL staining and caspase 3 activity. Other cells were transplanted into the spleens of Wistar rats with a 90% hepatectomy 12 hours later. RESULTS: Western blot analysis and RT-PCR confirmed the expression of hBcl-2 in AdCMVhBcl-2-infected hepatocytes. Recombinant FasL produced a dose-dependent increase in TUNEL-positive percentage and caspase-3 activity in uninfected hepatocytes, but did not influence these features in AdCMVhBcl-2-infected cells. On challenge with 90% hepatectomy, the survival of Wistar rats receiving transplantation of AdCMVhBCL-2-infected hepatocytes was significantly prolonged compared with the controls. CONCLUSION: Adenovirus-mediated BCL-2 gene transfer protects transplanted hepatocytes from Fas-mediated cytolysis, thus holding promise for a new avenue of acute liver failure treatment.     

Hepatocyte transplantation through the hepatic vein: a new route of cell transplantation to the liver

The efficiency of hepatocyte transplantation into the liver varies with the method of administration. This study investigated whether retrograde infusion via the hepatic vein provides a sufficient number of donor cells for the liver. Donor hepatocytes were isolated from dipeptidyl peptidase IV (DPPIV(+)) rats and transplanted into DPPIV(-) rat livers either by antegrade portal vein infusion or retrograde hepatic vein infusion. Hepatocyte engraftment ratios and localization were evaluated by histological DPPIV enzymatic staining at 1 week and 8 weeks after the transplantation. No significant differences in engraftment efficiency were observed at either 1 week or 8 weeks after transplantation by either route. However, the localization of the transplanted hepatocytes differed with the administration route. Portal vein infusion resulted in predominantly periportal engraftment, whereas hepatic vein infusion led to pericentral zone engraftment. Immunohistochemical analysis showed that the transplanted hepatocytes engrafted in the pericentral zone after retrograde infusion displayed intense CYP2E1 staining similar to the surrounding native hepatocytes. CYP2E1 staining was further enhanced by administration of isosafrole, an inducing agent for various cytochrome P450 enzymes, including CYP2E1. This study demonstrates a novel approach of transplanting hepatocytes into the liver through retrograde hepatic vein infusion as the means to target cell implantation to the pericentral zone.