One-Year Chromaffin Cell Allograft Survival in Cancer Patients With Chronic Pain: Morphological and Functional Evidence

Cell Transplantation, Vol. 7, No. 3, pp. 227–238, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0963-6897/98 $19.00 1 .00 PII S0963-6897(97)00161-9 Original Contribution ONE-YEAR CHROMAFFIN CELL ALLOGRAFT SURVIVAL IN CANCER PATIENTS WITH CHRONIC PAIN: MORPHOLOGICAL AND FUNCTIONAL EVIDENCE JEAN C. BÉS,* JEAN TKACZUK,* KIMBERLY A. CZECH,† MATHIEU TAFANI,* RAYMOND BASTIDE,* CLAUDE CARATERO,* GEORGE D. PAPPAS,† AND YVES LAZORTHES*1 *Laboratory of Pain and Cell Therapy, Faculty of Medicine, University Paul Sabatier, 133 route de Narbonne, Toulouse 31062, France, and †Department of Anatomy & Cell Biology, University of Illinois at Chicago, Chicago, IL 60612 M Abstract — The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-b-hydroxylase (DbH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space. © 1998 Elsevier Science Inc. ble pain in terminal cancer patients. However, these routes of morphine administration introduce problems related to mechanical malfunctions of tunneled catheters, injection ports, and implantable pumps as well as an increased risk of infection (12,13). These drawbacks in using morphine pumps have triggered research in finding alternative therapies for treating severe pain conditions (26,36). The implantation of adrenal medullary chromaffin cell tissue into the subarachnoid space of the spinal cord has been shown to provide stable relief of chronic pain that surpasses the conventional use of morphine in both animal and clinical trials [for review, see (4). The theoretical basis for this pain relief is thought to be the production and release of opioid peptides and catecholamines by chromaffin cells (20,22,24). When implanted, adrenal chromaffin cells release a “cocktail” of peptides that diffuse locally into the dorsal horn of the spinal cord where they modulate, and possibly block the transmission of somatosensory information sent to higher brain centers. Although the mechanism of pain relief with adrenal medullary implants has not been thoroughly characterized, several studies point to attenuation of NMDA receptor-mediated hyperalgesia as the primary action of chromaffin cell products (10,28). Because chromaffin cells release a combination of peptides including growth factors (epidermal growth factor, fibroblast growth factor, nerve growth factor), catecholamines (epinephrine, norepinephrine, and dopamine), and other proteins (enkephalins and endorphins) (30,31), it is probable that all of these products contribute M Key Words — Neural transplants; Adrenal medulla; Chromaffin cells; Cancer pain; Opioid peptides. INTRODUCTION Pharmacologic approaches that are currently available for the management of chronic cancer pain have certain limitations. Some patients rapidly develop tolerance to parenteral opioid peptides, necessitating progressively increasing dosages that often produce undesirable side effects. In most pain clinics intrathecal administration and/or intracerebroventricular administration of morphine is the therapy of choice to combat severe intractaACCEPTED11/26/97. 1 Correspondence should be addressed to Yves Lazorthes, M.D., Laboratory of Pain and Cell Therapy, Faculty of Medi- cine, University Paul Sabatier, 133 route de Narbonne, Toulouse 31062, France. 227 228 Cell Transplantation ● Volume 7, Number 3, 1998 either directly or indirectly to the analgesic effect of chromaffin cell implants. A series of studies by our laboratory at The University of Illinois at Chicago showed that chromaffin cells from both allogeneic and xenogeneic sources can release catecholamines and opioid peptides into the CSF, and they reduce pain sensitivity in well-known chronic pain paradigms (8,9,23,35). Interestingly, no immunosuppressant or antiinflammatory agents were required in any behavioral studies performed with rodents using allografts. Supporting the validity of this methodology, healthy TH-positive implants of adrenal medullary tissue have been shown in the spinal cord several weeks following implantation (18). In addition, the survival period was reported as long as 6 mo for intrathecal allografts in rats based on increased CSF levels of opioid peptides and catecholamines compared to pretransplant levels (24). Our initial clinical experience at Toulouse, France, has indicated that immunosuppression with cyclosporin A (10 mg/kg) for not more than 2 wk after transplantation is apparently sufficient for long-term allograft survival and pain reduction (7,14,15). Immunosuppression is required, at least initially, for animals implanted with the chromaffin cells obtained from xenogeneic sources (18). More recently, experiments with xenogeneic chromaffin cells that are encapsulated using a semipermeable polymer membrane (without immunosuppression) have defined a new methodology that has proven functional in animal, preclinical, and clinical trials (1,3,11,25). These studies have also shown that the released products from chromaffin cell implants can significantly reduce pain sensitivity for limited periods of time and have corroborated the findings using unencapsulated allogeneic tissue. The efficacy of adrenal medullary implants for the treatment of chronic pain has been demonstrated in rodents by several groups, using different pain paradigms, and have provided a sound foundation for initiating clinical trials in humans. The first application of these cellular properties of chromaffin cells as a therapeutic strategy for controlling cancer pain in humans was reported by the Vaquero group (32,33). The results from this study were considered unsatisfactory. Histological findings indicated necrotic reduction in 50% of the grafted tissue. A second series of five patients underwent trials of the same nature in our preliminary study at the University of Illinois at Chicago. However, tissue was maintained in culture for several days before transplantation to insure viability with more positive results showing the reduction of both morphine intake and pain scores in four of the patients (26,36). Unfortunately, autopsy permission was not obtainable so the viability and integration of grafted tissue fragments were not confirmed by histology. However, CSF samples taken periodically throughout the study showed Met-enkephalin and catecholamine levels at several magnitudes higher than pretransplant levels. A further study using the same procedure with eight patients at Toulouse University Hospital (France) was recently reviewed (14), and this data was combined with the clinical studies at The University of Illinois Medical Center (36). Similar findings were reported by both groups that showed that 10 out of 13 patients benefited from adrenal medullary implants and, in some patients, morphine intake ceased whereas in other cases it was reduced and, more importantly, it had stabilized. If chromaffin cell therapy is proposed as an alternative to such traditional approaches to pain relief, as chronic intrathecal morphine administration after failure of oral therapy, it is important to establish its long-term efficacy in humans. In this study we report on the clinical and pharmacotherapeutic findings in two terminal cancer patients who were followed for 12 mo after receiving adrenal chromaffin cell tissue transplants. Graft function was assessed using subjective measures, such as pain scores, and objective measures such as morphine intake, CSF levels of Met-enkephalin, and histology of grafted tissue obtained from autopsy. Our results show that both patients benefited from transplants of adrenal medullary implants with a significant reduction in pain scores. In addition, biochemical assays and histological studies indicate that chromaffin cells were present in grafted tissue and producing catecholamines and enkephalins. In summary, our results indicate that adrenal medullary transplantation has the potential to provide significant analgesia for as least 1 yr in cancer patients suffering from chronic pain. MATERIALS AND METHODS Patient Selection This study was carried out at Toulouse University Hospital (France) after examination and approval of the protocols by the Committee of Ethics and People Protection (CCPPRB). Patient selection included informed consent and both participants in the study presented with identified progressing cancerous lesions associated with chronic pain that initially responded well to oral morphine but, because of irreversible side effects, oral intake was replaced by intrathecal administration. Ambulatory follow-up was possible in both reported cases. Tissue Preparation Adrenal medullary glands were obtained from male organ donors who tested negative for viral serologies HIV-l and 2, HTLV-l and 2, and hepatitis B/C. Following dissection from the kidneys, glands were placed in Chromaffin cell allograft and cancer pain ● J.C. BÉS cold Belzer liquid and transferred to the laboratory. The duration of the protocol for isolation of adrenal medullary tissue from cortical tissue, modified from Winnie et al. (36), did not exceed 2-1/2 h. During the isolation procedure, each gland was maintained in a media solution (Dulbecco’s modified Eagle’s—DMEM—and Ham’s F12—1:1 DMEM: F12) supplemented with antibiotics (penicillin/streptomycin, 100 U/mL; gentamicin. 50 mg/mL; kanamycin, 25, mg/mL), antifungicide (Fungizone) (amphotericin [B, 0.125 mg/mL), and 50% FBS (Source of supplies: GibcoBRL, Life Technologies, France). Adrenal medullary tissue was dissected from cortical tissue with microscissors under binocular loop (Olympus), cut into small pieces of 0.5 to 2.0 mm3 in size and cultured at standard conditions (37°C with 5% CO2) in media containing 50% fetal bovine serum (FBS). During the course of a week, the percentage of FBS in media was gradually reduced and adrenal medullary pieces were maintained in culture at standard conditions with media containing 10% FBS until needed for transplantation. Grafts of adrenal chromaffin cells were performed on the eighth day following isolation of adrenal medullary tissue for the first patient (P1), on the 10th day for the second (P2). In both cases, a sample of tissue was taken for morphological analysis at the light microscopic level prior to transplantation to confirm viability of the graft tissue. Light microscopic analysis was assessed by immunoreactivity of chromaffin cells in tissue pieces to antityrosine hydroxylase (TH) and/or antidopamine-bhydroxylase (DbH) markers on frozen sections as described previously (18). Neurochemical analysis of the culture media for Met-enkephalin was also performed to provide chemical support of chromaffin cell secretion from the graft tissue and to parallel the morphological findings. Met-enkephalin levels were determined by radioimmunoassay using a commercially available kit (Methionine Enkephalin 125 RIA kit, Incstar, Sorin, France). Met-enkephalin levels were determined using standard radioimmunoassay procedures (5). Transplantation Procedure and Patient Follow-Up The graft procedure and the follow-up of patients were performed according to previously described methods (14,19). In brief, an injection access Miniport (Cordis) was situated in both cases on the level L4 –L5, of which the distal end of catheter was respectively at T10 for P1 and T9 for P2. Then, a small CSF sample was obtained for measuring normal CSF levels of Met-enkephalin to establish a baseline control for future comparisons. Then, approximately 1 mL of adrenal medullary tissue suspended in a balanced salt solution (without FBS) was carefully infused into the CSF of the lumbar cistern. ET AL. 229 For P1, two grafts were performed at different times with adrenal glands taken from two male donors, 40 and 35 years old, respectively. The dissection product was limited because of the dysmorphia of the first donor’s glands, and the availability of only one gland for the second donor (the second gland was inadvertently destroyed by the surgeon). Following transplantation of the first adrenal medullary graft, P1 was immunosuppressed for a period of 15 days with cyclosporin A (l0 mg/kg daily, Sandoz): P1 was not immunosuppressed after the second graft. For P2, two grafts were also performed: donors were male 23 and 42 years old, respectively. In the first donor, only the right gland was used because the left gland was hypotrophied. Medullary tissue was very sparse in the second donor’s glands and difficult to separate from cortical tissue. No immunosuppressive treatment was used following transplantation of the second adrenal medullary allograft. The implanted access port was kept in place for the duration of the study, and this allowed for periodic sampling of CSF for monitoring Met-enkephalin levels without subjecting patients to the pain and discomfort from repeated lumbar punctures. CSF examination started on the day of the graft (day 0) with a second sample taken 8 days later (day 8) and then periodically every month subsequent to obtaining these controls. Cytological examinations of CSF were performed the routine way, on Nageotte’s cell after cytocentrifugation. Two histological slides were stained with Maygrunwald Giemsa, four slides were fixed with acetone at 220°C and saved for eventual immunocytochemical tests. Concentrations of Met-enkephalin were measured by radioimmunoassay as described above. At day 0, pain intensity was determined in both using a visual analog scale (VAS, where 0 5 no pain and 10 5 the most severe pain) and behavioral evaluation was carried out according to the Karnofsky scale. After transplantation, patient-reported pain scores were noted on a monthly basis. Autopsy Graft tissue was removed 8 h after P1’s death and 12 h after P2’s. Autopsy was limited to the dorsal spinal axis and cauda equina. After laminectomy, a large exeresis of the dural sheath and its contents was performed between T7 and sacrum, after ligature and section of the roots at the internal orifice of the intervertebral foramen. Autopsy specimens were ligated at their upper portion. After fluid was aspirated for cytological analysis, specimens were then put into IX HlSTOChoicey Tissue Fixative (Amresco, Solon, OH) with 20% ethanol. After opening the dura mater and examination with a surgical microscope (OPMI-l, Zeiss), the implant access ports 230 Cell Transplantation ● Volume 7, Number 3, 1998 were noted and photographed before the removal of the grafts. Histological Study Dopamine-b-Hydroxylase (DbH) Immunostaining. Following fixation of tissue, 1 mm3 cell blocks were cut from the transplant, embedded in paraffin, and 10 mm sections were cut and placed on slides. Tissue sections were deparaffinized and rehydrated using standard methods. Slides with sections were placed in xylene for 5–10 min, dipped in a series of alcohol solutions for 3 min each, then followed by phosphate-buffered saline (PBS) for 15 min. Endogenous peroxide activity was removed by a 40% methanol, 3% hydrogen peroxide solution in PBS for 20 min. Nonspecific binding sites on tissue sections were then blocked with 5% donkey serum (Amersham) in PBS-BSA (PBS with 0.8% bovine serum albumin, BSA) for 30 min. After washing three times (5 min) with PBS-BSA, sections were incubated overnight (4°C) in DbH (lncstar, diluted 1:1000). The following day, sections were rinsed in PBS-BSA three times (5-min intervals), followed by incubation in secondary antirabbit biotinylated antibody (Amersham, 1:200 dilution) for 60 min at 4°C. Sections were washed again in PBS-BSA three times (5 min), streptavidin biotinylated horseradish peroxidase complex (Amersham, 1:100 dilution) was added, and incubated for 30 min at 4°C. Sections were rinsed in PBS three times (10 min), reacted with DAB (3,39-diaminobenzidine, Sigma) for 1–10 min, dehydrated with a series of alcohol solutions, followed by xylene, and mounted with permount. Tyrosine Hydroxylase (TH) Staining. Following fixation with HISTOChoicey, areas of the spinal cord containing transplanted adrenal chromaffin cell pieces were dissected and placed in 20% sucrose solution in 0.1% phosphate buffer overnight at 4°C for cryoprotection. Cryostat sections of isolated tissue were cut at 10 –12 mm, mounted on slides, and incubated overnight at 4°C with TH monoclonal antibody (1:500 dilution, lncstar). Preimmune serum instead of primary antibody was used on control sections. Next day, sections were rinsed three times in PBS followed by incubation for l h (at room temperature) with rhodamine-linked secondary antibody (1:100 dilution, Cappel). After mounting on slides and coverslipped with Fluoromount, sections were observed through an Olympus BX6O epifluorescence microscope. Chromagranin A Immunostaining. A technique similar to the one described above (avidin– biotin–peroxidase) was performed on deparaffinized paraffin sections for chromogranin A (lncstar, diluted 1:300) immunoreactivity. RESULTS Clinical and Biological Data Patient 1. The first patient (P1), who had been operated on 6 yr previously, presented with lung cancer localized in the lower right lobe. Lateral-thoracic, homolateral pain occurred subsequent to neoplastic pleural invasion. This pain, which failed to respond to different therapies, was partially controlled (VAS, 3–5) for a year with buprenorphine (Temgesic three times/24 h). Slowrelease morphine (Moscontin) was then prescribed but had to be discontinued, despite much significant analgesic action, because of severe and irreversible digestive troubles. After 5 yr of chronic evolution, pain gradually became more disabling (VAS between 9 –10), limiting this patient’s autonomy and he subsequently required permanent assistance (Karnofsky index: 50). Because of the side effects with morphine taken orally, intrathecal administration of morphine (2.5 mg/24 h, single dose) was advised and this provided good, but incomplete analgesia (VAS, 3–5) without the associated digestive complaints common to oral morphine. Morphine therapy through intrathecal administration provided adequate pain relief initially, but with time P1 developed tolerance and the chronic pain associated with his condition returned (VAS 7– 8). Because no other alternative pain therapies were available to provide relief, P1 consented to participate in the study to see whether a transplant of adrenal medullary chromaffin cells could alleviate his chronic pain. The first intrathecal graft of about 1 mL of adrenal medullary tissue was performed 4 mo later following patient consent and committee approval for admission into the study. Preparation conditions were fulfilled (measurement of Met-enkephalin in culture media at 2500 pg/mL) as described, and the patient was given cyclosporin A (according to the protocol, 10 mg/kg 24 h, Sandoz) as a precautionary measure to preclude any risk of graft rejection. On day 8, analgesia was excellent (VAS 2) and it was noted that this patient’s general condition had improved (Karnofsky index 70); however, he still required intrathecal morphine, which had stabilized at the initial dose. After 15 days, cyclosporin A treatment was stopped because the patient complained of nausea. Despite a significant increase in CSF Met-enkephalin levels and a reduction in pain scores, a second graft was performed on day 135 in an attempt to decrease morphine intake. Again, about 1 mL of adrenal medullary tissue pieces suspended in a balanced salt solution was Chromaffin cell allograft and cancer pain ● J.C. BÉS injected into the lumbar cistern through the implanted access port. The measurement of Met-enkephalin, from supernatant media obtained from cultured adrenal medullary tissue to be used as the second graft, revealed basal secretion at 2800 pg/mL. The patient was not given any immunosuppressive treatment following the second graft procedure. Results over a period of 360 days are shown in Fig. 1. Pain scores (Fig. 1A, P1) go from 8 to 2 at the end of the first week and remained stable at this level. After the second graft, pain levels dropped to between 0 and 1 and functional activity remained excellent until the eighth month (Karnofsky index .70) when P1’s general condition quickly worsened with pain occurring again. The patient was maintained at 2.5 mg/24 h of morphine for 190 days and then weaned to 1.0 mg/24 h by 1 120 days (Fig. 1B, P1). During the terminal period, however, the dosage was increased to 3 mg/24 h over 30 days. After a period of increased Met-enkephalin in CSF (Fig. 1C, P1) between day 0 (80 pg/mL) and day 190 (320 pg/mL), variations occurred with lower values until day 1210 (between 120 and 150 pg/mL), with gradual increase during the last 3 mo (around 200 pg/mL). Death occurred on day 1385. Patient 2. The second patient (P2) was operated on for infiltrating cancer of the right urethra (enlarged nephrectomy followed by radiotherapy). He presented with subsequent progressive pain in the bilateral lumbar area and in the right perineal and crural areas. At first the pain responded well to slow-release morphine (100 mg/24 h), but after 6 mo treatment, morphine tolerance developed and efficacy was under 50% with undesirable digestive side effects when doses were increased. As this patient’s general condition worsened (Karnofsky index 5 50) and pain became more severe, he was placed on intrathecal morphine at 10 mg/24 h . This allowed for greater pain relief without the side-effects associated with higher oral doses of morphine. This pain therapy provided relief for the patient for only 2 mo before his condition worsened and the pain returned (VAS 8 –9). Because intrathecal morphine administration is the option that provides the most potent pain relief and this therapy was inadequate, P2 consented to be a participant in this study to see if an adrenal medullary transplant could alleviate the chronic cancer pain that had been unyielding to intrathecal morphine therapy. A graft of about 1 mL adrenal medullary tissue pieces was performed into the lumbar cistern through an access port. Prior to transplantation, a sample of culture medium was obtained from adrenal medullary tissue pieces maintained in culture, and this revealed a rate of Metenkephalin at 2600 pg/mL. ET AL. 231 Following transplantation, P2’s general condition had improved (Karnofsky index 5 70), as demonstrated by excellent pain control from day 18 to day 130 (VAS 0.5–1), and this finding was complemented by reduced morphine doses that had stabilized to 2.5 mg/24 h. But because of renewed pain, doses of daily morphine had to be progressively increased to 12 mg/24 h between day 160 and day 190. Therefore, a second graft was performed on day 1136. The graph in Fig. 1 shows the results obtained for both adrenal medullary grafts in P2 over a period of 360 days where pain scores (Fig. 1A, P2) are reduced from 9 to 1 after the first week and are between 0 and 1 on day 130; afterwards, they increase until day 1120 (VAS 5) and appear to stabilize after the second graft (VAS, 0 –1) until day 1330. Biochemical data on P2 shows that Met-enkephalin levels in CSF (Fig. 1C, P2) showed two peaks on day 130 and day 1150 (respectively at 270 and 300 pg/mL) from initial values of about 100 pg/mL; Table 1 and Fig. 1C contains all CSF neurochemical data. Although Met-enkephalin levels fell, after an initial peak, within 3 mo of receiving the first transplant, following a second transplant at day 1136, high levels were maintained and appear to stabilize for the subsequent 8 mo until P2’s death. During the terminal period, perineal lumbar pain and the radix pain of the inferior right member were completely alleviated. There was only distal dysesthesia of inferior members related to lymphedema subsequent to pelvic extension of the cancer, which did not respond well to opioids. Analgesic control remained excellent (Fig. 1A, P2) until the patient’s death (day 1362), despite no increases in morphine doses throughout the follow-up period (Fig. 1B, P2). Morphological Analysis Autopsies. Access ports are generally situated between T12 and the sacrum, but one was located at the level of T9 in P1 (Fig. 2A). In both cases, access ports were located at points where isolated nodular formations (0.2– 0.3 mm in diameter) and, as was typically the case, multilobulated nodules of irregular size (up to 10 mm length) could be seen (Fig. 2B). These formations were located along thoracic and lumbosacral roots of the cauda equina (Fig. 2C) and seemed to adhere to the radicular sheath and were without visible macroscopic extension into the roots. Only one nodular location was found on the internal face of the dura mater on the level of the external foramen of the posterior root (Fig. 2D), whereas filum terminale and conus medullaris were spared in both cases. On the whole, injected material spread proximally from the injection area at the level of lumbar enlarge- 232 Cell Transplantation ● Volume 7, Number 3, 1998 Fig. 1. (A) Patient-reported pain score (from 0 5 no pain to 10 5 the most intense pain). P1: patient 1; P2: patient 2; Single arrow 5 initial chromaffin cell graft; double arrows 5 second transplant. (B) The complementary daily intrathecal morphine intake (mg/24 h). (C) The Met-enkephalin release into the CSF (pg/mL). The mean basal level of CSF Met-enkephalin was done for 10 nongrafted patients (hatched areas). This was the reference value for the CSF Met-enkephalin rate of grafted patients. Chromaffin cell allograft and cancer pain ● J.C. BÉS ET AL. 233 Table 1. The complementary intrathecal morphine intake (mg/24 h) and the CSF Met-enkephalin release (pg/mL) data Following (days) J0 18 130 160 190 2,5 2,5 2,5 1 1 1 1 1 3 3 3 3 170 280 320 150 160 130 120 — 210 230 200 190 5 2,5 12 12 12 10 12 12 12 12 12 12 270 210 105 110 300 280 280 260 250 280 190 210 P1 Intrathecal morphine 2,5 2,5 (mg/24 h) Met-enképhaline/CSF 80 130 (pg/mL) P2 Intrathecal morphine 10 5 (mg/24 h) Met-enképhaline/CSF 100 230 (pg/mL) 1120 ment and conus medullaris and distally into the area of dural pouch without precise systematization between the two areas. The graft pieces furthest from the injection sites were found at 18 cm, but because the anatomic elements studied did not contain higher cervicodorsal spine and brain, we could not exclude further locations. Histology Nerve roots did not present demyelination or segmentary degeneration at the level of the grafts. They did not adhere to the radicular sheath and were irregularly limited by a thin encapsulating connective tissue lamina. A fibroconnective tissue structure that sometimes developed between the fragments of adrenal medullary tissue pieces constitutes an important polymorphic conglomerate in both patients. Rare mononuclear infiltrates were observed in P2’s biopsies; however, no inflammatory cells could be seen in P1’s graft. In some sections, microscopic examination revealed some necrosis involving a part of the implanted tissue. The chromaffin granules appeared dense and dispersed throughout the cytoplasm, (Fig. 5), while large areas of degranulation can be observed with characteristic vacuolization with the electron microscope [cf. (16)]. With the light microscope, the structural heterogeneity of antichromagranin A tissue immunoreactive areas (Fig. 3) confirms the neuroendocrine specificity of these nodular formations (i.e., the grafted tissue). In the same way, positive reaction to enzymatic markers (TH and DbH) reveals a functional identification of chromaffin cells (Fig. 4). DISCUSSION Several animal studies have provided substantial evidence supporting the use of adrenal chromaffin cell implants as a potentially useful therapy for chronic pain (8,23,36). These studies show that implants of chromaffin cells to the subarachnoid space can consistently reduce pain sensitivity in such well-established chronic pain paradigms as the adjuvant-induced arthritic rat 1150 1180 1210 1240 1270 1300 1330 1360 model (23) and the Bennett-Xie model (8). From previous biochemical studies, the mechanism of pain reduction is postulated to occur by the release of natural pain-relieving substances such as catecholamines and, especially, by opioid peptides that are coreleased (20,22). These neuroactive substances secreted from chromaffin cells travel locally through the subarachnoid space and diffuse into the dorsal horn of the spinal cord where primary sensory afferents synapse. It is hypothesized that the secreted products of chromaffin cells either modulate the synaptic function of somatosensory afferents by inhibiting synaptic activity or by activating local sensory afferent inhibitory circuits. Most recent studies suggest that the mechanism of pain relief in chromaffin cell implants may be through inhibition of the NMDA receptor-mediated events in secondary afferent neurons (10,28). Based on these animal studies that have characterized the mechanism and efficacy of pain relief with chromaffin cell implants in rats, our group received permission by ethics committees to initiate preliminary clinical trials on human patients with chronic intractable pain resulting from terminal cancer. The collaborative study between the Toulouse group at the University of Paul Sabatier and the Chicago group at The University of Illinois at Chicago has shown that 11 out of 15 cancer patients benefited from chromaffin cell allografts (19). If chromaffin cell transplantation is proposed as an alternative therapy to the conventional use of intrathecal morphine, it is important to establish the long-term efficacy of chromaffin cell grafts in humans. The purpose of this article was to report on two of the patients with longterm chromaffin cell allografts that survived and functioned for 1 yr. Transplant function in patients was assessed using several subjective and objective analyses that included patient-reported pain scores, morphine intake, CSF levels of Met-enkephalin, and histological staining of transplant tissue obtained at autopsy. For subjective analysis of transplant function, P1 and P2 rated the severity of their pain on the Visual Analog 234 Cell Transplantation ● Volume 7, Number 3, 1998 Fig. 2. Autopsy specimens from P1 show the dispersion of grafted tissue fragments (A, whole spinal cord, reduced, ruler length 16 cm) from the injections site (L4/L5). Note (arrows), multilobulated nodules of irregular size (B), clusters of adrenal medullary tissue between the roots of the cauda equina (C), and one nodular location in the inner aspect of the dura mater (D) (mag: B, 33; C, 43; D, 43). Scale (VAS) where 1 represented the least and 10 the most intense. Pain scores reported by both patients showed that within the first week following implantation of adrenal medullary tissue there was a rapid decrease in pain scores from 7 to 2 in P1, and 8 to 1 in P2. Essentially, in the course of 1 wk, P1’s and P2’s perception of pain changed from severe pain to having mild or no pain. Besides pain scores, additional evidence that adrenal chromaffin cell allografts are functional for pain relief is based on biochemical studies showing that CSF levels of Met-enkephalin increased following Chromaffin cell allograft and cancer pain ● J.C. BÉS Fig. 3. Low-power photomicrograph (A) of a section through two fragments of adrenal medullary tissue isolated from the lumbar-sacral roots and immunostained for chromagranin A (CGA) reactivity. At higher magnification (B), CGA-positive remnants revealed the rounded morphology of chromaffin cells and cellular and collagenous elements (arrowheads) between these nodular formations (mag: A, 3203; B, 22003). transplantation. Synthetic opioids have well-known pharmacological applications in the treatment of severe acute and chronic pain symptoms (37,38). The natural analog to morphine are enkephalins, which are produced by chromaffin cells and coreleased with catecholamines (17). In both patients (P1 and P2), normal CSF levels of Met-enkephalin prior to transplantation were 80 and 100 picograms, respectively. Following the first transplant, Met-enkephalin levels increased to 170 picograms (130 days) in P1 and 270 picograms (130 days) in P2. This demonstrated a high correlation to pain scores (which fell from 10 to 1 and 10 to 2, respectively, in patients P1 and P2). After the second transplant, Met-enkephalin levels reached as high as 320 picograms in P1 (at 190 days) and 300 picograms in P2 (at 1 150), which correlated again to reduced pain scores. Although Met-enkephalin is only one of many opioid peptides believed to be secreted by chromaffin cells, this biochemical assay has provided evidence that chromaffin cells are probably releasing many other neuroactive substances (30,31). Further- ET AL. 235 Fig. 4. Low-power photomicrograph (A) of dopamine-b-hydroxylase (DbH)-stained section through the graft site. (B) Chromaffin cells immunohistochemically stained with tyrosine hydroxylase (TH) antibody with a rhodamine-linked secondary antibody marker. Note the intensity of TH labeling. (mag: A, 1603: B, 12003). more, the high correlation of CSF Met-enkephalin levels to patient pain scores indicates that opioid production by chromaffin cells is a major factor Fig. 5. The electron microscopy study (330,000) show an important tissue alteration with degenerative chromaffin cells in necrotic zone but also the evidence of some secretory granuli. 236 Cell Transplantation ● Volume 7, Number 3, 1998 responsible for pain relief. Among the other released products from chromaffin cells that may contribute to pain relief are several neurotrophic factors including fibroblast growth factor (FGF), transforming growth factor, and ciliary neurotrophic factor (CNTF) (31). Recent reports suggest that a diminution of neurotrophic factors may contribute to the pathophysiology of certain neuropathies, and artificial replacement strategies of growth factors have been shown to reverse peripheral neuropathy [for review, see (2)]. In both cases, the decision to give a second transplant was based either on a modest increase in Met-enkephalin levels or when pain scores/morphine intake increased. Therefore, a second transplant was performed on P1 at day 140 and on P2 at day 1136 after the first transplant. Following a second transplant, patient P1 was relatively pain free for the next 330 days, which corresponded to only a slight increase in morphine intake. In patient P2, a second transplant resulted in pain relief for an additional 230 days but with no reduction in morphine intake. It should be noted in both cases that morphine intake was stabilized and patient Met-enkephalin levels in CSF were double and close to triple their baseline value prior to transplantation. It is believed that the apparent weaker analgesic effect with the first transplant in both patients was due to an inadequate amount of adrenal chromaffin tissue transplanted. We have found that about 2 mL of small, viable adrenal medullary tissue fragment (0.5–2.0 mm3 in size) should be transplanted initially to obtain optimal results so that additional transplants may not be necessary (36). The problems associated with obtaining enough healthy human donor tissue is not uncommon, and has sparked interest in alternative donor sources such as xenografts (1,6). The difficulty faced with obtaining human adrenal medullary tissue presents a unique problem for several reasons. Although adrenals are routinely removed from cadaver donors together with the kidneys, it can be difficult obtaining glands with a large amount of intact adrenal medullary tissue. Also, a thick adrenal medulla is needed because some chromaffin cells/tissue are lost during microdissection and culture. In addition, because of the awkward position of the adrenals when cadavers are supine and their close proximity to the kidney, many surgeons fear that donor kidneys may be injured while trying to dissect the adrenal gland from the perirenal fascia. Therefore, often only a fraction of the adrenal tissue may be obtained instead of the entire gland. Both patients, P1 and P2, died as a result of their terminal cancer condition at 1385 days and 1362 days, respectively. Prior to death, consent was obtained permitting autopsy to assess graft survival and morphological changes, if any, of the grafted and host tissue. However, the tissue was not preserved with fixative until 8 h (in P1) and 12 h (P2) following death, so it was recognized that cellular morphology would not be in pristine condition. Gross morphology of the spinal cord and nerves in the lumbar cistern and in rostral spinal segments indicated that there was postimplantation migration. In P1, adrenal tissue fragments were identified as high as T6 and as low as S1. This migration of tissue may reflect the response of tissue fragments to gravity as a result of the sedentary behavior of P1 and P2. However, the histology of implants from our rodent studies have not indicated that there is migration of graft because adrenal medullary tissue appears to be firmly attached to the pia mater of the dorsal spinal cord and in close proximity to the implantation site (23). This is not surprising if one considers that, because human and rodent donor adrenal medullary tissue pieces are of similar size (1 mm3), the lumbar cistern in our clinical trials is a much larger space and can accommodate more movement of tissue than in the rat subarachnoid space. Transplant survival in patients P1 and P2 was assessed by immunostaining for specific chromaffin cell markers in grafted tissue fragments from autopsy specimens. This reveals that cells in the graft were tyrosine hydroxylase, dopamine-b-hydroxylase, and chromagranin A positive. The presence of these enzymes confirms that chromaffin cells were a component of the tissue fragments, and they were producing catecholamines providing morphological evidence of graft function in P1 and P2. Biochemical and morphological analyses of graft function are evidence that adrenal medullary implants into the subarachnoid space can control pain in cancer patients for at least 1 yr. Patient pain scores decreased drastically in both cases. A reduction in pain scores corresponded to an increase in CSF Met-enkephalin levels that were double and triple their preimplantation level. Furthermore, the morphology of implanted adrenal medullary tissue fragments indicated that chromaffin cells were implanted and they were actively producing and secreting catecholamines (19). Despite the fact that no effort was made to crossmatch donor and recipient, there appeared to be no rejection of implanted chromaffin cells. Infiltrating leukocytes were absent in graft fragments and glial tissue around it. The adrenal medullary tissue graft appeared intact. In summary, the use of implanted adrenal medullary tissue represents a new approach to treating cancer pain that rivals current pharmacological approaches such as implantable morphine pumps, which are expensive and have many side effects. 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