Macroautophagic process was differentially modulated by long-term moderate exercise in rat brain and peripheral tissues.

The autophagic process is a lysosomal degradation pathway, which is activated during stress conditions, such as starvation or exercise. Regular exercise has beneficial effects on human health, including neuroprotection. However, the cellular mechanisms underlying these effects are incompletely understood. Endurance and a single bout of exercise induce autophagy not only in brain but also in peripheral tissues. However, little is known whether autophagy could be modulated in brain and peripheral tissues by long-term moderate exercise. Here, we examined the effects on macroautophagy process of long-term moderate treadmill training (36 weeks) in adult rats both in brain (hippocampus and cerebral cortex) and peripheral tissues (skeletal muscle, liver and heart). We assessed mTOR activation and the autophagic proteins Beclin 1, p62, LC3B (LC3B-II/LC3B-I ratio) and the lysosomal protein LAMP1, as well as the ubiquitinated proteins. Our results showed in the cortex of exercised rats an inactivation of mTOR, greater autophagy flux (increased LC3-II/LC3-I ratio and reduced p62) besides increased LAMP1. Related with these effects a reduction in the ubiquitinated proteins was observed. No significant changes in the autophagic pathway were found either in hippocampus or in skeletal and cardiac muscle by exercise. Only in the liver of exercised rats mTOR phosphorylation and p62 levels increased, which could be related with beneficial metabolic effects in this organ induced by exercise. Thus, our findings suggest that long-term moderate exercise induces autophagy specifically in the cortex.

Coracoid bone block versus arthroscopic Bankart repair: a comparative paired study with 5-year follow-up.

INTRODUCTION AND HYPOTHESIS: The hypothesis of this study was that the rate of recurrence of anterior instability of the shoulder after arthroscopic Bankart repair with suture anchors is higher than after coracoid bone block (Latarjet procedure). MATERIALS AND METHODS: This continuous retrospective monocentric study included a cohort of patients who underwent surgery for post-traumatic recurrent antero-inferior instability (2004-2005): 51 patients who underwent an open Latarjet procedure were paired for age at surgery to 51 patients who underwent an arthroscopic Bankart repair. All patients were evaluated with a questionnaire and 50% were evaluated in a follow-up consultation with X-rays. Recurrent instability was defined by at least one episode of anterior dislocation or subluxation. RESULTS: Demographic data, soft tissue and bone lesions were statistically similar between the groups. At 5 years follow-up, the recurrence rate was 24% in the Bankart group and 12% in the Latarjet group (P=012). In the Bankart group, age under 25 years old (P=0.01), competitive sports after surgery (P=0.01), and glenoid erosion (P=0.02) were independent risk factors of recurrence. In the Latarjet group, five technical errors were identified out of six cases of recurrence. Fifteen of the 18 cases of recurrence did not undergo revision surgery because patients remained satisfied with their results. DISCUSSION AND CONCLUSIONS: At 5 years of follow-up, the rate of recurrent instability following arthroscopic Bankart repair was two times higher than that following the coracoid bone block procedure. Young patients who wish to practice a competitive sport or present with glenoid erosion are poor candidates for arthroscopic Bankart repair. The rate of recurrence is extremely high in unselected patients. The open Latarjet procedure results in a fairly high rate of recurrence due to technical errors. LEVEL OF EVIDENCE: Level IV (retrospective study).

Role of matrix metalloproteinase-9 (MMP-9) in striatal blood-brain barrier disruption in a 3-nitropropionic acid model of Huntington's disease.

AIMS: 3-Nitropropionic acid (3-NPA) is a natural toxin that, when administered to experimental animals, reproduces the brain lesions observed in Huntington's disease, which mainly consist of selective neurodegeneration of the striatum. The lesions also include severe alterations to the blood-brain barrier (BBB), which increase its permeability to several substances including blood components and exogenous fluorescent dyes, and the concomitant degradation of some of its constituents such as endothelial cells, tight junction proteins and the basement membrane. We studied here the role of matrix metalloproteinases (MMPs)-2 and -9, also called gelatinases A and B, in the degradation of the BBB in the striatal lesions induced by the systemic administration of 3-NPA to Sprague-Dawley rats. METHODS: 3-NPA was intraperitoneally administered at a dose of 20 mg/kg once a day for 3 days. MMPs were studied by means of immunohistochemistry and in situ zymography. RESULTS: In 3-NPA-treated rats, MMP-9 was present in most of the degraded blood vessels in the injured striatum, while it was absent in vessels from non-injured tissue. In the same animals, MMP-2 staining was barely detected close to degraded blood vessels. The combination of MMP-9 immunostaining, in situ zymography and inhibitory studies of MMP-9 confirmed that net gelatinolytic activity detected in the degraded striatal blood vessels could be attributed almost exclusively to the active form of MMP-9. CONCLUSION: Our results highlight the prominent role of MMP-9 in BBB disruption in the striatal injured areas of this experimental model of Huntington's disease.

Can we improve the indication for Bankart arthroscopic repair? A preliminary clinical study using the ISIS score.

The objectives of this study on arthroscopic treatment of chronic anterior shoulder instability were the collection of the current practices for this indication, their development as reported in the literature, and the analysis of preliminary results on a multicenter prospective series of Bankart arthroscopic procedures undertaken using a common technique on patients selected based on the Instability Severity Index Score (ISIS). This procedure predominates in the English-speaking world, whereas the Latarjet protocol is preferred in France. The choice between the two seems to be cultural since neither technique could be demonstrated to be superior in an analysis of 171 responses to an Internet questionnaire in this study. The literature reports disappointing results in the Bankart arthroscopic procedure and recent articles have researched the predictive factors for its failure. Eleven centers prospectively included 125 patients from 1 December 2007 to 30 November 2008. The inclusion criteria were recurrence of anterior instability and an ISIS less than or equal to four points out of 10. All the selected patients underwent capsuloligamentous reinsertion with a common minimal technique of at least three anchors and four sutures with the same postoperative protocol. At a mean follow-up of 18 months, four patients (3.2%) had experienced recurrence. For the 84 patients reexamined at 1 year, the Walch-Duplay and Rowe scores were, respectively, 88.4 and 87.8 points out of 100. Subjectively, 88.1% of the patients declared they were satisfied and would undergo the intervention again. This study confirmed the use of the ISIS as a consultation tool. Only continuation of the study with a minimum follow-up of 3 years will allow us to validate the lower limit of the ISIS below which this technique could be proposed provided that it respects the technical prerequisite of at least four capsuloligamentous sutures.

Recurrence of femoral echinococcosis 5 years after a primary surgical procedure.

We report a case of recurring femoral hydatid disease in a trisomic-21 patient 5 years after a primary surgery. The patient presented a thigh abscess with a lateral supracondylar area fistula. The workup demonstrated massive osteolysis involving the proximal, diaphyseal,and distal femur as well as multiple soft tissue cystic masses but no metastases. Treatment consisted in cystic masses debridment, extensive bony-curettage of the intraosseous cystic zones, temporary weight-bearing suppression, and albendazole. The patient remains under follow-up and cannot be considered definitely cured.

Surgical management of elbow dislocation associated with non-reparable fractures of the radial head.

BACKGROUND: The "terrible triad" of the elbow is the combination of an elbow dislocation, radial head and a coronoid process fracture. Because of a combined sagittal, frontal and transverse instability, these injuries are notoriously difficult to treat. We report our results with a technique for reconstruction of "terrible triad" injuries with either no facture or a type I fracture of the coronoid process in addition to a non-reparable radial head fracture. The hypothesis of this study was that standard surgical treatment of this lesion using a "deep to superficial" stabilisation by a single lateral approach and radial head replacement enables early and reliable functional results. PATIENTS: From June 2004 to January 2007, 13 patients with an average age of 40 years at the date of trauma (range 18-77) underwent reconstruction of a "terrible triad" injury of the elbow with the same technique. The mean follow-up was 25 months (range 15-48). RESULTS: Eighty-four percent of the patients were very satisfied and satisfied. Average flexion was 131 degrees (110-140). Average extension was -11 degrees (-30-0). Average pronation was 72 degrees (40-80). Average supination was 70 degrees (50-80). The grip strength averaged 75% of that of the non-injured side (50-105). All elbows were stable at review. Eight complications occurred. CONCLUSION: Our results suggest that some terrible triad injuries can be successfully managed with deep to superficial stabilisation by lateral approach, consisting in three-dimensional stabilisation done by anterior capsular reinsertion with absorbable anchors, radial head replacement and lateral collateral ligament repair. This standard management provides enough stability to allow early active rehabilitation, preventing post-operative instability and stiffness. This procedure appears to be reliable and reproducible.

[Percutaneous osteosynthesis of lumbar and thoracolumbar spine fractures without neurological deficit: surgical technique and preliminary results]. / Ostéosynthèse percutanée des fractures lombaires et thoracolombaires non neurologiques: technique chirurgicale et résultats préliminaires.

PURPOSE OF THE STUDY: The aim of this work was to study the technique of percutaneous osteosynthesis of lumbar and thoracolumbar spine fractures without neurological deficit and to report preliminary results. MATERIAL AND METHODS: This retrospective study included 15 patients with lumbar or thoracolumbar spine fractures who were treated between January 2004 and January 2006 by percutaneous osteosynthesis. There were seven men and eight women, mean age 36 years (range 16-58 years). The Magerl classification (AO) was A1 (n=4), A2 (n=1), A3 (n=9), B2 (n=1). Levels were T12 (n=1), L1 (n=10), L2 (n=2), L3 (n=1), L4 (n=1). A specific instrument set was used to insert a short fixation using two pedicular screws on either side of the fractured vertebra and two prebent 5.5mm rods introduced with an aiming device. The operation was performed under fluoroscopy. Ten patients wore a removable corset. The upright position was allowed if there were no other injuries. Computed-tomography scans were obtained preoperatively, postoperatively and at two years follow-up. Function was assessed with the Oswestry score. RESULTS: Mean operative time was 108 minutes (range 40-180 minutes). None of the patients with an isolated spinal injury required blood transfusion. Mean hospital stay was 12 days (range 4-28). Results were expressed for 13 patients whose operations were exclusively percutaneous. Mean follow-up was 17 months (range 6-30). The visual analog scale (VAS) was 1.6/10. The mean Oswestry score was 16. Three quarters of the patients resumed their occupational activities. None of the patients was dissatisfied. Mean vertebral kyphosis (VK) improved from 16 to 8.1 degrees , corrected regional angle (CRA) from 12 to 2.5 degrees at last follow-up. Loss of correction at last follow-up was 1.1 degrees for VK and 2.5 degrees for CRA. The rate of pedicle screw malposition was 3.8%. There were no cases of disassembly nor material failure. There were no infections. None of the implants had to be removed. DISCUSSION: Percutaneous osteosynthesis of the spine is technically feasible, but requires considerable experience. Functional and subjective results have been good. The loss of correction at last follow-up has been comparable to that observed with conventional open surgery. This technique is an intermediary method between orthopaedic treatment and conventional surgery. Exact indications must be established. CONCLUSION: Percutaneous osteosynthesis of lumbar and thoracolumbar spine fractures is an attractive therapeutic option. Our results are encouraging. Indications and limitations of this technique must be carefully identified.

[Fence grafting for acetabular reconstruction with a cementless cup]. / Greffe en palissade pour recentrage acétabulaire dans les reprises par implant sans ciment.

Acetabular reconstruction is difficult after loss of bone stock and socket remodeling. Several techniques have been proposed ranging from a metal backing to allografting. We propose fence grafting. After explantation, the acetabulum is carefully cleaned of all interface tissue and precisely measured. If the vertical diameter is clearly greater than the anteroposterior diameter, a tricortical graft is harvested from the iliac crest and modeled to perfectly fit between the anterioinferior iliac spine and the residual posterior wall as well as the fundus medially. One or two oblique screws are inserted for stabilization. Any superior bone loss is filled by bone substitute (without mechanical value). The acetabulum is then reamed from the obturator foramen sparing the anterior and posterior columns. Residual bony defects are filled with cancellous bone. A hemispheric cup is then press fit and maintained with two or three screws. We performed this procedure in eight patients with SO.F.C.O.T. stage III acetabular loosening with segmentary bone loss and an oval acetabular cavity. Clinical follow-up was more than four years. The Postel-Merle-d'Aubigné score improved from 9.8 to 15.7 on average. Radiographically, there were no implant mobilization or migration and no circumferential lucent lines were observed. A nearly anatomic position was achieved in all cases except two (technical imperfection). At more than one-year follow-up, the grafts could not be distinguished from adjacent bone. For us, high-positioned or jumbo cups do not offer a satisfactory reconstruction option. There is a risk of compression with allografts from a head bank. We have not used the cemented metal-backed solution nor impacted grafts. The major drawback with fence grafting is the iliac harvesting (possible residual limping because of the extensive disinsertion of the gluteus medius. The reliable acetabular reconstruction is the major advantage. This technique is not simply an acetabular block widened laterally but it decreases the vertical dimension. This is a reliable but minute technique which allows true long-lasting reconstruction of the acetabulum.

Abnormalities in dendritic cell and macrophage accumulation in the pancreas of nonobese diabetic (NOD) mice during the early neonatal period.

Dendritic cell (DC), macrophage (Mphi) and lymphocyte infiltrations have been observed in normal human perinatal pancreata, but have never been investigated so early in control mice. In type 1 diabetes-prone NOD mice, these cells are thought to infiltrate first the periphery of the islets of Langerhans around weaning before further islet infiltration and beta-cell destruction. We quantified, during the first month of life, the numbers of DC (characterized by CD11c positivity and dendritic morphology), histiocyte-like Mphi (characterized by ER-MP23 positivity) and Mphi with scavenging potential (characterized by BM8 positivity) in C57BL/6, DBA/2 and BALB/c control, and NOD and lymphocyte-deficient NODscid mouse pancreata. First, CD11c+ DC were present at low densities from birth onwards in control pancreata, while densities were higher in NOD and NODscid. Second, high numbers of BM8+ and ER-MP23+ Mphi were observed at birth in all strains investigated. After birth, particularly BM8+ cells disappeared progressively in control strains, but not in NOD and NODscid. Third, NOD mice also had more ER-MP23+ Mphi at birth compared to controls. Finally, DC and Mphi localizations were similar in all strains, i.e., mostly as dispersed cells in perivascular, periductular, peri-islet areas and interlobular septa. The most remarkable finding was that particularly BM8+ Mphi, were seen at sites of islet neogenesis and predominantly at the duct-islet interface. Our data showed that different types of APC were present in the pancreas during postnatal development in various control mouse strains and some differences were observed in NOD and NODscid mice from birth onwards.

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells.

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.

Islet endocrine-cell behavior from birth onward in mice with the nonobese diabetic genetic background.

BACKGROUND: Glucagon-producing alpha cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate alpha cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes. MATERIALS AND METHODS: We determined alpha-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward. RESULTS: NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (<2,000 pixels) as C57BL/6 mice. During the postnatal period, the percentages of glucagon-positive areas in islets less than 2000 pixels were higher in NOD mice than C57BL/6; only a trend was found in NODscid. Pancreatic mRNA expression and glucagon content decreased in all strains at weaning. However, before weaning, pancreatic and blood glucagon levels were significantly lower in NOD and NODscid compared to C57BL/6 mice. Low basal nonfasting glycemia was observed in all strains before weaning with some strain differences: glycemia was significantly lower in NOD than C57BL/6, and higher in NODscid than NOD and C57BL/6. CONCLUSION: These data suggest that, before weaning, NOD and, to some extent NODscid pancreata contain more immature islets (as reflected by their small size and high percentages of glucagon-positive areas, concomitant with lower glucagon storage and basal secretion) than C57BL/6 pancreata.

Immunohistochemical study of lymphoid tissues in adjuvant arthritis (AA) by image analysis; relationship with synovial lesions.

The aim of this study was to examine leucocyte populations in lymphoid organs during AA and to ascertain the relationship with lesions in synovial joints. Popliteal lymph nodes, spleen and knee synovial membranes were removed from both healthy and AA rats at intervals of 3-4 days over a 3-week period. Cryostat sections were stained with MoAbs directed against lymphocyte and macrophage subpopulations, and studied by image analysis. Throughout the arthritic period, high numbers of ED1+ and ED3+ macrophages were seen in both lymphoid compartments and intercellular adhesion molecule-1 (ICAM-1) expression also increased in some zones of lymph nodes and spleen. The percentages of CD4+ and CD8+ cells rose in the splenic zones studied but fell in the lymph node cortex. Very few natural killer (NK) cells were found in lymphoid tissues, but the number rose after AA induction. In synovia from AA rats, ED2+ macrophages proliferated but alpha/beta T cell infiltration was only occasionally observed, accompanied by ED1+ cells and ICAM-1 expression. In conclusion, synovitis developing after AA induction seems to be caused directly by macrophages and indirectly by lymphocytes placed both in popliteal lymph nodes and spleen.

An image analysis strategy to determine the distribution of cell types in spleen sections.

An image analysis strategy was designed to objectively determine distribution patterns of cell types in spleen sections. The strategy was applied to rat spleen cryostat sections that were strained immunohistochemically by means of monoclonal antibodies against different populations of lymphocytes and macrophages. The strategy revealed three segments of the spleen beginning in the middle of a central arteriole and ending within the red pulp. In each of these segments, three consecutive zones were established: the white pulp, the marginal zone, and the red pulp. In each tissue section, three segments were selected starting in two different arterioles. Consequently, six segments were analysed in each section. Special software was used to calculate percentages of positive staining in all zones in each segment. Image analysis data for each monoclonal antibody tested correlated closely with microscopical observations. The proposed strategy allows objective quantification of lymphocyte and macrophage populations and their distribution patterns. It is an useful tool for studying imbalances in cell populations in the spleen due to immune challenges.

Alterations of lymphocyte populations in lymph nodes but not in spleen during the latency period of adjuvant arthritis.

The aim of this study was to analyze potential imbalances in lymphocyte populations from regional lymph nodes (LN) and spleen occurring before the development of the outer inflammation of adjuvant arthritis (AA). Percentages and absolute numbers of CD5+, CD4+, CD8+, Ig+, I-A+, NKR-P1+ and TCRgammadelta+ cells were determined. No differences in percentages of gammadelta T or NK cells were found either in LN or spleen, thus ruling out an important role of these minor subpopulations in these early stages of AA. While no significant lymphocyte imbalances were observed in spleen, an increase in the percentage of B lymphocytes was found in regional LN. Moreover, a high proliferation of CD8+ cells was observed when measuring absolute numbers of LN lymphocytes, thus producing an imbalance in the CD4/CD8 ratio at very early stages of the inflammatory process. These findings suggest a role for CD8+ and B lymphocytes in the latency period of AA at the LN level. Our results indicate a primary role for lymph nodes in initiating the inflammation of AA, whereas cells from the spleen probably play a secondary role.

Kinetics of W3/25 anti-rat CD4 monoclonal antibody. Studies on optimal doses and time-related effects.

Although anti-CD4 monoclonal antibodies (MoAb) have been proven successful in preventing or treating adjuvant arthritis, little is known about the duration of the effects of these MoAb and their pharmacokinetics. In this work, we report the effects of a mouse anti-rat CD4 MoAb, named W3/25, on peripheral blood lymphocytes from female Wistar rats. Animals received a single dose of W3/25, from 1 to 3 mg, and blood was sampled at different time points from 0 h to 15 days after MoAb administration. After erythrocyte lysis, samples were stained by indirect immunofluorescence and analyzed by flow cytometry. Pharmacokinetic data were studied by assessing plasma levels of mouse IgG1 by ELISA-sandwich. W3/25 produced the down-regulation of surface CD4 molecule as early as 20 min after its administration at doses of 2 and 3 mg. The same effect was seen 30 min after a dose of 1 mg. The recovery of lymphocytes with normal expression of CD4 also depended of the dose administered. Thus, CD4+ lymphocytes were recovered at 48, 72 and 96 h in rats treated with 1, 2 or 3 mg of W3/25, respectively. Plasma levels of free antibody were detectable from 20 min to 72 h, 60 min to 48 h and 60 min to 24 h after administration of 3, 2 and 1 mg, respectively, of W3/25. The mouse IgG1 MoAb used in this study followed a two-compartment model and its behavior was linear.

Treatment with an anti-CD4 monoclonal antibody strongly ameliorates established rat adjuvant arthritis.

Some experimental arthritic diseases can be prevented by treatment with anti-CD4 MoAbs. Trials with ongoing disease have not been successful so far. The aim of this study was to ascertain whether W3/25 could reverse adjuvant arthritis (AA), when beginning treatment on day 14, i.e. when the disease was established. Moreover, one group of animals treated with the anti-CD4 MoAb received OX8 MoAb at the same time, thus depleting CD8+ cells from circulation. During treatment with W3/25, a strong amelioration of inflammatory signals were observed, as assessed by means of paw volume increase and arthritic score. However, when treatment stopped, a rebound to arthritis signals occurred. The parallel depletion of CD8+ cells did not modify these effects, thus the combined treatment W3/25 + OX8 gave the same amelioration as treatment with W3/25 alone. These findings indicate that CD4+ cells play an important role in perpetuating rat AA. Moreover, CD8+ cells do not seem to have a regulatory role int he CD4+ cells responsible for the inflammatory response.

Depletion of gamma/delta T cells does not prevent or ameliorate, but rather aggravates, rat adjuvant arthritis.

OBJECTIVE: To investigate the role of gamma/delta T cells in Mycobacterium tuberculosis-induced rat adjuvant arthritis. METHODS: Rats with adjuvant arthritis were injected with anti-T cell receptor gamma/delta (anti-TCRgamma/delta) monoclonal antibody V65 according to a preventive protocol, a pre-arthritis peak protocol, and a late therapeutic protocol. Arthritis severity and joint destruction were monitored, and depletion of target cells was analyzed by flow cytometry. RESULTS: Although all protocols led to successful depletion of TCRgamma/delta(bright) cells in peripheral blood and lymph nodes, none of the regimens influenced clinical parameters of adjuvant arthritis. If rats were treated before the clinical peak of adjuvant arthritis, however, joint destruction was significantly more severe than in vehicle-treated rats. CONCLUSION: Rat adjuvant arthritis is not promoted or perpetuated by gamma/delta T cells. Aggravation of joint destruction with pre-arthritis peak anti-gamma/delta treatment suggests a stage-dependent protective role of gamma/delta T cells in adjuvant arthritis.

Comparison of four lymphocyte isolation methods applied to rodent T cell subpopulations and B cells.

The aim of this study was to establish the validity of four lymphocyte isolation methods. The effects of three different erythrocyte lysing methods commonly used in the analysis of human cells, namely, lysis by ammonium chloride (AC), Becton Dickinson lysis (BDL) and the Coulter Q-Prep (CQP) preparation system were established by flow cytometry on rat lymphocyte subsets. The results were compared with those obtained with a Ficoll-Isopaque (FI) density gradient procedure adapted for use with rat cells. Lymphocyte isolation by AC or FI gradient was performed before labelling the lymphocyte subpopulations, whereas the BDL and CQP methods were performed after staining the cells in whole blood. The FI gradient yielded the lowest CD5+, CD4+ and CD25+ cell percentages. On the other hand AC lysis produced higher percentages of T cells and lower percentages of B cells than the other methods studied. The percentages obtained after BDL or CQP methods for T lymphocyte subsets and B cells were found to be reproducible. The commercial methods (BDL and CQP) are faster but rather expensive, whereas AC lysis and FI gradient separations are cheap and particularly useful when there is a requirement to culture the cells.