Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome.

To determine the survival of cultured allogeneic keratinocytes transplanted to a deep dermal bed 24 tattoos that had been removed by deep shave excision in 19 patients were grafted with sheets of cultured allogeneic keratinocytes from donors of the opposite sex. Cells carrying the Y chromosome were identified in biopsy specimens taken from the graft site by in situ DNA hybridisation with a biotinylated Y probe (pHY 2.1) and visualised with a technique using immunoperoxidase. The cultured allograft sites were biopsied one, two, and three weeks after transplantation. No male cells were identified in any biopsy specimen from female patients who were given transplants of male cultured keratinocytes, and all biopsy specimens from male patients, who received female cultured keratinocytes, showed percentages of male cells within the normal range for male skin. The beneficial effects of cultivated allogeneic keratinocytes result from effects on wound healing other than forming a successful graft that "takes."     

Long-term survival of human skin allografts in patients with immunosuppression

Severe burn patients lack adequate skin donor sites to resurface their burn wounds. Patients with severe burn injuries to areas such as an entire face are presently reconstructed with skin grafts that are inferior to normal facial skin. This study was designed in part to determine whether human skin allografts would survive, repopulate, and persist on patients with immunosuppression and after discontinuation of immunosuppression. Small split-thickness skin grafts were synchronously transplanted at the time of renal transplantation from six renal transplant donors to recipients. All six patients were immunosuppressed with the usual doses of renal transplant immunosuppressants (methylprednisolone, cyclosporine, prednisone, and azathioprine). The skin allografts were biopsied when rejection was suspected and at various intervals. Special histologic studies were performed on skin biopsy specimens. Class II DNA tissue typing was performed on transplanted and autogenous skin biopsy specimens of four patients. Fluorescent in situ hybridization was performed successfully on skin biopsies of four patients' transplanted skin and on two of these four patients' autogenous skin. All six human skin allografts sustained a 100 percent take and long-term clinical survival. DNA tissue typing performed on skin allograft biopsy specimens from patients taking immunosuppressants all revealed donor and recipient cells. DNA tissue typing performed on autogenous skin biopsies from the same patients all revealed only recipient cells. Fluorescent in situ hybridization performed on allograft and autogenous specimens from patients taking immunosuppressants revealed transplanted donor cells with rare recipient cells in the allograft and only recipient cells in the autogenous skin. This study of six patients proves that it is possible for human skin allografts to survive indefinitely on patients taking the usual dosages of immunosuppressants used for renal transplantation. There was minimal repopulation of skin allografts by autogenous keratinocytes and fibroblast while patients were taking immunosuppressants. Immunosuppression was discontinued in two patients after renal transplant rejection after 6 weeks and 5 years. When immunosuppression was discontinued after 5 years in one patient, the skin allograft cells were destroyed and replaced with autogenous cells, but the skin graft did not reject acutely and persisted clinically. It is hypothesized that the acellular portion of the skin allograft was not rejected acutely because of relatively low antigenicity and because it acted as a lattice for autogenous cells to migrate into and replace rejected allograft skin cells. No chimerism was seen in autogenous skin in the skin-renal transplant patients in this study.     

Clinicopathologic studies on human epithelial autografts and allografts.

We compared the survival of cultured epithelial allografts and epithelial autografts applied to donor sites for split-thickness skin grafts. Before grafting, cultured epithelium was devoid of Langerhans cells (LCs) or lymphoid cells by immunohistochemical and electron microscopic examinations. The autografts attached to the wounds permanently, without any clinical evidence of rejection. In contrast, allografts, which were mismatched for MHC and blood-type antigens, appeared to adhere firmly only until day 7. By the second week, signs of graft rejection were apparent: The graft changed color, and the underlying dermis underwent "microerosion" and denudation. By the third week, the area formerly occupied by the allograft had the same coloration as ungrafted wounds and apparently had undergone reepithelialization by the host. Immunohistochemical and ultrastructural studies clearly demonstrated that host Langerhans-like cells (without Birbeck granules) appeared in both autografts and allografts. However, these cells were numerous and distributed widely throughout allografts, whereas they were scarce and confined to the basal layer of autografts. Typical Langerhans cells (containing Birbeck granules) were present in the prickle-cell layer of autografts by day 7. The present study strongly indicates that allografts of cultured epithelium are rejected. Furthermore, given the known ability of Langerhans-like cells to function as accessory cells in T-cell activation, our results point to a role for host Langerhans-like cells in immunologically mediated rejection of the epithelial allografts.     

Prolongation of skin xenograft survival with modified cultured fibroblasts

Cyclosporine A, one of the most potent immunosuppressive drugs, mediates some of its immunosuppressive and nephrotoxic effects by enhancing transforming growth factor-beta secretion and receptor expression. In this experimental study, the effect of cyclosporine pretreatment of cultured dermal fibroblasts on xenogeneic tissue rejection after microimplantation beneath skin grafts was investigated. The effects of the site-specific immunosuppressive strategy on skin xenograft survival were tested. Because the skin is an immunological indicator of the rejection of composite tissue allografts, it was considered that this strategy could be used as a supportive therapy for composite tissue allotransplantation in the future. In the first stage of the study, fibroblast cultures obtained from skin biopsy samples from five rats were treated with different single doses of cyclosporine (100 to 3000 ng/ml), and transforming growth factor-beta levels were measured in culture supernatants after 72 hours. In the second stage, 60 Sprague-Dawley rats were divided into six groups, as follows. For group I (sham), only the standard grafting procedure was performed. For group II, after the standard grafting procedure, rats were treated with intraperitoneal injections of 30 mg/kg (n = 5) or 10 mg/kg (n = 5) cyclosporine for 10 days. For group III, cultured fibroblasts obtained from skin biopsy samples from rats were treated with 100 or 500 ng/ml cyclosporine, and the cells were collected by light trypsinization and centrifugation after 72 hours. After the standard skin grafting procedure, modified fibroblasts were implanted between the graft and the recipient bed with a Pasteur pipet. For group IV, the same procedures as for group III were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. For group V, in addition to standard grafting, unmodified fibroblasts (not treated with cyclosporine) were implanted between the graft and the recipient bed. For group VI, the same procedures as for group V were performed and then rats were treated with 10 mg/kg cyclosporine, administered intraperitoneally, for 10 days. The rejection process was observed macroscopically, and statistical significance was determined with the Mann-Whitney test (p < 0.01). Graft survival times were significantly prolonged in groups III and IV, compared with groups I, II, V, and VI (p < 0.001). No difference between groups III and IV was observed. The novel finding of this investigation is that xenogeneic skin graft survival times could be prolonged with microimplantation of cyclosporine-treated cultured fibro-blasts.     

The use of allogeneic cultivated keratinocytes for the early coverage of deep dermal burns – indications, results and problems

Since 1995, keratinocytes are grown into cultures and used as allografts for the coverage of deep dermal defects in our burn unit. Donor skin samples are mostly acquired from other burn patients. In addition, special methods of skin preservation allow us the use of skin, which has been taken in redundancy for split thickness skin grafting from nonburned patients.Thirty five patients with deep partial thickness burns in the face were treated since 1996 according to the following concept: Dermabrasion or tangential excision was performed before the 5(th) day following trauma. If viable dermis was present, the wounds were covered with sheets of allogeneic cultivated keratinocytes. In cases of deeper defects, autologous skin grafts were applied. In 23 cases, epithelialisation was achieved within 10 days, in 8 patients, a prolonged duration until complete healing was observed. In 5 faces, coverage of residual defects with skin grafts was necessary. The mentioned problems of wound healing occurred from infection, incomplete excision of burn eschar and a depth of the wound which was retrospectively seen too deep for the treatment with keratinocytes. At follow up, patients were examined clinically and functionally with Frey's faciometer(R), which is an instrument for quantification of mimic movements. In cases of uncomplicated healing, a nearly complete restitution was found.Other indications include deep dermal burns in children and the coverage of early excised wounds in adults, with a reasonable amount of viable dermis remaining, both resulting in a significant reduction of donor-site morbidity. In severely burned adults with limited donor sites, it offers the possibility of immediate definite coverage of large areas.     

Clinical application of autologous cultured epithelia for the treatment of burn wounds and burn scars

This report presents our experience with autologous cultured human epithelia grafting on burn wounds, burn scars, and skin-graft donor sites in seven patients. Dispersed epidermal cells were cultured with 3T3 cells treated with mitomycin C. After 2 to 3 weeks, cultured epithelia (total 350 to 2250 cm2) were grafted to the wound. The results showed that cultured epithelia grafts did not take so completely compared to the meshed skin grafts used for the coverage of burn wounds. However, cultured grafts placed on aseptic wounds adhered well and showed good appearance. In the histologic findings, normal differentiation of epidermal cells was found. Cultured grafts were bordered from subepidermal granulative tissue with basement membrane. A rete ridge and the adnexal structures were absent in the specimens that adhered to the burn wounds. However, in the specimens that took on abraded wounds, a gently sloping rete ridge and elastic fibers were seen. The histologic findings showed structures resembling normal skin.     

Leukotoxin,a linoleate epoxide: Its implication in the late death of patients with extensive burns

Burn death based on circulatory shock is often encountered after recovery from primary shock in patients with deep and extensive burns,i.e., late death. Several toxic substances have been proposed, however, the responsible substance remains obscure. Since we have found leukotoxin, a highly cytotoxic linoleate epoxide biosynthesized by neutrophils, in the burned skin, in the present study we determined plasma leukotoxin concentrations in various degree of 30 burn patients. C-reactive protein and circulatory white blood cells were also measured. A significantly high mortality rate of patients with extensive burns (burn surface area over 70%) was observed compared with that in patients with burn surface area under 70%, and significantly high leukotoxin concentrations were observed within a week, and 3 weeks after the thermal injury in patients with extensive burns compared with those in patients with burn surface area under 70%. There were two peaks of plasma leukotoxin concentrations,i.e., the early phase (within 1 week) and the late phase (over 1 week) in patients with extensive burns. Plasma leukotoxin concentrations significantly correlated with burn surface area in the early phase, and similar correlations were observed in the late phase. A significantly high mortality rate (61%) of patients with peak leukotoxin concentrations over 30 nmol/ml was observed compared with 8% for those below 30 nmol/ml. Plasma leukotoxin concentration correlated significantly to C-reactive protein concentration, log (leukotoxin nmol/ml)=0.042×C-reactive protein (mg/dl)+0.74, (r=0.83,P<0.01) in the late phase. From these results, it is concluded that leukotoxin is produced in patients with burns particularly in the late phase of extensive burns, and leukotoxin might play an important role in the tissue destructive procedure associated with severe burns.     

Lymphocyte and blast cell glomerulonephritis in renal allografts of rats injected with phytohaemagglutinin

Rejection of Ag-B incompatible skin allografts on skin pedicles was delayed until the third week after grafting. The pedicles were confirmed to be alymphatic since there was no proliferative response in the ipsilateral lymph nodes as assessed by ³H-thymidine incorporation after ip injection. However, a variable proliferative response was noted in the spleen when allografted rats were compared to isografted rats. Surprisingly, most of the labeled cells were situated in the red pulp and germinal centers; relatively few proliferating cells were seen in the thymus-dependent areas. If the proliferation of host T cells is necessary for the rejection of alymphatic skin grafts, its location remains uncertain.     

Early excision and grafting of face and neck burns in patients over 20 years

Although excision and grafting of burns has become common and standard, many surgeons have been reluctant to excise and graft face burns. In fact, we could find photographic results at 1 year after grafting of only eight patients in the English literature. We began excision and grafting of face burns in 1979 and presented our first 16 patients in 1986 in this journal. With encouragement from Janzekovic and Jackson, we continued and have now used essentially the same procedure for more than 20 years in approximately 100 patients and, from this large series, are able to present outcomes. From January of 1979 to May of 1999, we performed excision and grafting on 91 patients with deep face burns. Data were recorded and 35-mm photographs were obtained throughout the 20-year period. We reviewed that database and the slide files of these patients. We found 45 patients with complete photographic sets including 1-year follow-up. Since, in our opinion, there is no useful, objective measure of appearance, we decided to simply publish all 45 sets of complete photographs, permitting the reader to subjectively form an opinion of the outcome of this procedure. The results are all shown as "full" face burns and two "partial" face burns. We continue to believe that early excision and grafting is indicated for face burns that will not heal within 3 weeks and that the procedure yields results that permit the burn victims to return to society and minimizes the time off work or out of school.     

削痂植皮术后结合负压封闭引流在深度烧伤患者中的应用效果及对血清致痛因子及炎性因子的影响

摘要 目的：探讨削痂植皮术后结合负压封闭引流在深度烧伤患者中的应用效果及对血清致痛因子及炎性因子的影响,以此为临床治疗深度烧伤患者提供参考。方法：选取暨南大学附属第一医院在2018年1月至2022年1月期间收治的75例深度烧伤患者进行回顾性分析,所有患者均接受削痂植皮术治疗;按术后不同换药方法分为常规换药组和VSD组,其中常规换药组35例,术后常规换药;VSD组40例,术后采用VSD治疗。比较两组患者首次植皮成活率,术后1周、2周创面愈合率,创面愈合时间,疼痛程度及并发症发生率等,测定两组患者血清致痛因子、冲洗液炎性因子表达水平。结果：VSD组首次植皮成活率95.00%（38/40）,常规换药组首次植皮成活率71.43%（25/35）,差异有统计学意义（P<0.05）。VSD组术后1周、2周创面愈合率高于常规换药组,创面愈合时间、创面疼痛评分低于常规换药组,差异有统计学意义（P<0.05）。两组术后1周相关致痛因子表达较术前明显下降（P<0.05）,且VSD组致痛因子表达低于常规换药组,差异有统计学意义（P<0.05）。两组术后1周冲洗液炎性因子表达低于术前（P<0.05）,且VSD组冲洗液炎性因子表达与常规换药组比较下降明显,差异有统计学意义（P<0.05）。VSD组术后并发症发生率12.50%（5/40）低于常规换药组40.00%（14/40）,差异有统计学意义（P<0.05）。结论：削痂植皮术后结合负压封闭引流技术可提高深度烧伤患者创面愈合效果,增加首次植皮成活率,减少细菌生成、炎性因子的释放,减轻创面疼痛程度,值得临床进一步研究。     

Immunogenicity of nerve allografts in rats

The state of sensitization of rats after nerve or skin allograft was studied in vitro by using spleen cells or peripheral blood leukocytes (PBL) as responding cells and particulate alloantigen for stimulation, in culture conditions requiring or not supernatant from mixed lymphocytes culture. It is shown that 2-4 weeks after nerve or skin grafting a small number of PBL generates similar cytotoxic lymphocytic responses. However the state of sensitization induced by the nerve allografts is shorter compared to the one exhibited after skin allografts. On the other hand, no significant differences were observed at the level of the humoral response between both groups of grafted rats.     

Pinch skin grafting or porcine dermis in venous ulcers: a randomised clinical trial.

Chronic venous ulcers are common, and even with effective compression or elevation large ulcers may take months to heal. Pinch skin grafting may allow healing from epithelial islands throughout the surface area of the ulcer, and a prospective randomised trial was therefore conducted comparing this treatment with porcine dermis dressings. Most patients were treated as outpatients, 25 ulcers being randomised to treatment with pinch skin grafts and 28 to treatment with porcine dermis. Though the groups were well matched, the mean healing rate in the first week was 15 cm2 for pinch skin grafts compared with 3.5 cm2 with porcine dermis (p less than 0.02). By life table analysis 64% of ulcers treated by pinch grafts were healed at six weeks and 74% by 12 weeks compared with 29% and 46% of ulcers, respectively, treated with porcine dermis dressings (chi2 = 4.1; p less than 0.05). All ulcers that failed to heal within 12 weeks included an area posterior to the medial malleolus, where local compression may have been inadequate. Pinch skin grafting improves the rate of healing in large venous ulcers and is a simple technique that may be performed as an outpatient procedure under local anaesthesia.     

Cultured epidermal allografts: Quantitative evaluation of their healing effect in deep dermal burns

This study summarizes the Brno Burn Centre experience with the application of cultured epidermal allografts (CEAl) in the treatment of deep dermal burns. In a prospective randomised trial on 30 patients with deep dermal burns CEAl obtained from young healthy and examined donors and fixed on tulle grass carrier (Grasolind) were compared with empty Grasolind as the lowest layer of dressing. All the other layers were identical. Both kinds of dressing were applied simultaneously on the same deep dermal burn wound between 6th and 10th day after burn. Six days later the non-healed wound areas were recorded through painting on cellophane membrane and scanned in the computer. The percentage of wound reduction was calculated and statistically evaluated. The reduction of the non-epithelialized wound area was 86.5% when covered through CEAl and only 71.2% when covered with tulle grass (Grasolind) only. This difference is statistically significant. In conclusion it can be stated that cultured epidermal allografts strongly stimulate reepithelialisation in deep dermal burns. This revised version was published online in July 2006 with corrections to the Cover Date.     

Limb allografts in rats immunosuppressed with cyclosporine: as a whole-joint allograft

We performed limb allografts in three inbred rat strains immunosuppressed with cyclosporine. As controls, 10 autografts and 10 isografts exhibited an excellent result. In minor-mismatched allografts (Lewis to Fischer, n = 45), with the use of cyclosporine, the grafted limbs survived and the articular cartilage retained normal architecture and cell viability 52 weeks after grafting, but without cyclosporine treatment severe degeneration and destruction of articular cartilage were shown by 16 weeks after operation. In major-mismatched allografts (Brown Norway to Fischer, n = 35), the articular cartilage of cyclosporine-treated animals maintained normal architecture and cell viability 52 weeks after operation despite the gross appearance of skin rejection, while that of non-cyclosporine-treated animals was degenerated and destroyed by 6 weeks. These results suggest the possibility of whole-joint allografts in humans with the use of cyclosporine, as well as other organ transplantations.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Early excision and skin grafting of selected burns of the face and neck

Since 1979, 16 patients with facial and neck burns have been treated with excision and skin grafting within the first 4 days of injury. The injuries were tangentially excised and immediately covered with split-thickness skin grafts. Detailed consecutive results are presented. The patients can be divided into three groups. Group 1 consisted of small subdermal or circumscribed deep dermal burns of the face (n = 8). Healing was quick. Some patients developed signs of overgrafting. As a late result, unevenness and discoloration were seen. Group 2 consisted of mixed deep dermal and subdermal burns of the face and neck (n = 5). Usually, minor areas had to be regrafted. Some patients developed hypertrophic scars at border areas. In the completely excised and grafted area, the skin was smooth, pliable, and discolored. Group 3 consisted mostly of subdermal burns of the face and neck (n = 3). The surgical trauma was significant. Small areas had to be regrafted. Ectropion and microstomia developed. It is concluded that in selected cases of deep dermal and subdermal burns, early excision and skin grafting will result in faster healing and less scarring than expectant treatment.     

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2 h of aortic clamping and banked within 12 h of harvesting (13–14 h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.     

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider line arrange, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4°C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative,whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tissue engineering of cultured skin substitutes

Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin.     

Distribution of Neuropeptide Y-Immunoreactive Neurons in the Human Brainstem, Cerebellum, and Cortex During Development

1. Neuropeptide Y is found throughout the central nervous system where it appears to play a wide range of often poorly understood functions. In this study, the distribution of neuropeptide Y immunoreactive (NPY-ir) neurons in the brainstem, cerebellum, and cerebral cortex of human fetuses ranging in age from 11 gestational weeks to term was investigated by immunohistochemistry. 2. The NPY-ir cells were detected in the dorsal and ventral rostral midbrain and the interpeduncular nucleus by 21 weeks and 32 weeks of gestation, respectively. Although no positive cells were found in the pons, the NPY-ir fibers were detected there at 32 gestational weeks. 3. The vagal, hypoglossal, and olivary nuclei of the medulla oblongata contained immunoreactive cells by week 21 and the medullary reticular formation by week 25 of gestation. In most of these locations, both the number and size of neuropeptide Y positive cells were greater at birth and reached maximal values of 100-400 cells per 1 mm2 and 2-5 microm in diameter, respectively. 4. In the cerebellum, numerous NPY-ir horizontal and granule cells, as well as the cells within the dentate nucleus were observed as early as 21 weeks of gestation. 5. The NPY-ir cells were also detected in the developing cerebral cortex, with the earliest activity observed within the temporal cortex at 14 weeks of gestation. By week 21, positive cells appeared in the visual, frontal, sensory, and motor cortices. Most of these cells were bipolar or multipolar in morphology but their numbers at birth were relatively low. 6. Our results show a wide distribution of the NPY-ir cells in the developing human brain and offer supporting evidence for the important modulatory role of NPY in both the fetus and adult.