Evidence of an association between brain cellular injury and cognitive decline after non-cardiac surgery.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is common after non-cardiac surgery, but the mechanism is unclear. We hypothesized that decrements in cognition 1 month after non-cardiac surgery would be associated with evidence of brain injury detected by elevation of plasma concentrations of S100ß, neuron-specific enolase (NSE), and/or the brain-specific protein glial fibrillary acid protein (GFAP). METHODS: One hundred and forty-nine patients undergoing shoulder surgery underwent neuropsychological testing before and then 1 month after surgery. Plasma was collected before and after anaesthesia. We determined the relationship between plasma biomarker concentrations and individual neuropsychological test results and a composite cognitive functioning score (mean Z-score). RESULTS: POCD (≥-1.5 sd decrement in Z-score from baseline) was present in 10.1% of patients 1 month after surgery. There was a negative relationship between higher plasma GFAP concentrations and lower postoperative composite Z-scores {estimated slope=-0.14 [95% confidence interval (CI) -0.24 to -0.04], P=0.005} and change from baseline in postoperative scores on the Rey Complex Figure Test copy trial (P=0.021), delayed recall trial (P=0.010), and the Symbol Digit Modalities Test (P=0.004) after adjustment for age, sex, history of hypertension and diabetes. A similar relationship was not observed with S100ß or NSE concentrations. CONCLUSIONS: Decline in cognition 1 month after shoulder surgery is associated with brain cellular injury as demonstrated by elevated plasma GFAP concentrations.

Arterial pressure above the upper cerebral autoregulation limit during cardiopulmonary bypass is associated with postoperative delirium.

BACKGROUND: Mean arterial pressure (MAP) below the lower limit of cerebral autoregulation during cardiopulmonary bypass (CPB) is associated with complications after cardiac surgery. However, simply raising empiric MAP targets during CPB might result in MAP above the upper limit of autoregulation (ULA), causing cerebral hyperperfusion in some patients and predisposing them to cerebral dysfunction after surgery. We hypothesized that MAP above an ULA during CPB is associated with postoperative delirium. METHODS: Autoregulation during CPB was monitored continuously in 491 patients with the cerebral oximetry index (COx) in this prospective observational study. COx represents Pearson's correlation coefficient between low-frequency changes in regional cerebral oxygen saturation (measured with near-infrared spectroscopy) and MAP. Delirium was defined throughout the postoperative hospitalization based on clinical detection with prospectively defined methods. RESULTS: Delirium was observed in 45 (9.2%) patients. Mechanical ventilation for >48 h [odds ratio (OR), 3.94; 95% confidence interval (CI), 1.72-9.03], preoperative antidepressant use (OR, 3.0; 95% CI, 1.29-6.96), prior stroke (OR, 2.79; 95% CI, 1.12-6.96), congestive heart failure (OR, 2.68; 95% CI, 1.28-5.62), the product of the magnitude and duration of MAP above an ULA (mm Hg h; OR, 1.09; 95% CI, 1.03-1.15), and age (per year of age; OR, 1.01; 95% CI, 1.01-1.07) were independently associated with postoperative delirium. CONCLUSIONS: Excursions of MAP above the upper limit of cerebral autoregulation during CPB are associated with risk for delirium. Optimizing MAP during CPB to remain within the cerebral autoregulation range might reduce risk of delirium. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT00769691 and NCT00981474.

Should general anaesthesia be avoided in the elderly?

Surgery and anaesthesia exert comparatively greater adverse effects on the elderly than on the younger brain, manifest by the higher prevalence of postoperative delirium and cognitive dysfunction. Postoperative delirium and cognitive dysfunction delay rehabilitation, and are associated with increases in morbidity and mortality among elderly surgical patients. We review the aetiology of postoperative delirium and cognitive dysfunction in the elderly with a particular focus on anaesthesia and sedation, discuss methods of diagnosing and monitoring postoperative cognitive decline, and describe the treatment strategies by which such decline may be prevented.

Evaluation of two delirium screening tools for detecting post-operative delirium in the elderly.

BACKGROUND: Postoperative delirium in the elderly is common and associated with poor outcomes, but often goes unrecognized. Delirium screening tools, validated in postoperative settings are lacking. This study compares two screening tools [Confusion Assessment Method for the Intensive Care Unit (CAM-ICU) and Nursing Delirium Symptom Checklist (NuDESC)] with a DSM-IV-based diagnosis of delirium, conducted by neuropsychiatric examination in postoperative settings. METHODS: Consecutive English-speaking patients, ≥70 yr, undergoing surgery with general anaesthesia and capable of providing informed consent, were recruited. Diagnostic test characteristics were compared for each screening tool vs neuropsychiatric examination, both in the Post-Anaesthesia Care Unit (PACU), and daily during inpatient hospitalization, adjusting for repeated measures. RESULTS: Neuropsychiatric examination identified delirium in 45% of 91 patients evaluated in the PACU and in 32% of 166 subsequent delirium assessments on the ward in the 58 admitted patients. The sensitivity [95% confidence interval (CI)] of delirium detection of the CAM-ICU in the PACU, and in all repeated assessments was 28% (16-45%) and 28% (17-42%), respectively; for the NuDESC (scoring threshold ≥2), 32% (19-48%) and 29% (19-42%), respectively, and the NuDESC (threshold ≥1), 80% (65-91%) and 72% (60-82%), respectively. Specificity was >90% for both the CAM-ICU and the NuDESC (threshold ≥2); specificity for the NuDESC (threshold ≥1), in the PACU was 69% (54-80%) and 80% (73-85%) for all assessments. CONCLUSIONS: While highly specific, neither CAM-ICU nor NuDESC (threshold ≥2) are adequately sensitive to identify delirium post-operatively; NuDESC (threshold ≥1) increases sensitivity, but reduces specificity.

Hexaminolevulinate-mediated photodynamic purging of marrow grafts with murine breast carcinoma.

Photodynamic therapy (PDT) with porphyrin precursors is an established therapy for certain tumors. This study aimed to explore the use of hexaminolevulinate (HAL), a porphyrin precursor, for photodynamic purging of BM grafts contaminated with cells of the 4T1 breast carcinoma cell line. The optimal PDT dose was not effective in eradicating 4T1 cells when the tumor cells were mixed with murine marrow cells in vitro. However, the number of pulmonary metastases was reduced, and the survival of experimental animals was prolonged substantially when they were subjected to TBI followed by transplantation of syngeneic BM containing metastasized 4T1 cells that had been treated ex vivo by HAL-PDT. Despite the failure of in vitro experiments, HAL-based photodynamic purging could be a useful modality for treating animals bearing an experimental breast carcinoma.

Hexaminolevulinate-mediated photodynamic purging of leukemia cells from BM.

Photodynamic therapy (PDT) with porphyrin precursors has been established for tumor treatment. This study aimed at examining applicability of hexaminolevulinate (HAL) for photodynamic purging of leukemic cells from BM grafts and evaluating the clinical relevance of in vitro models. The PDT dose resulting in no colony formation by leukemic cells in vitro, in pure form or in a mixture with BM cells, was insufficient for complete killing of the leukemic cells ex vivo and for the treatment of the leukemia-bearing animals in vivo. The efficacy of HAL-PDT in cell lines in vitro should be verified in clinically relevant in vivo models.

Does intraoperative fluid management in spine surgery predict intensive care unit length of stay?

STUDY OBJECTIVE: To determine whether intraoperative fluid management in spine surgery predicts postoperative intensive care unit length of stay (ICU LOS). DESIGN: Retrospective case series. SETTING: University-affiliated medical center. PATIENTS: 103 adult ASA physical status I, II, and III patients undergoing spine surgery. INTERVENTIONS: Patients were divided into three LOS groups: no ICU stay (LOS0) (n = 26), 1 day ICU stay (LOS1) (n = 48), and ICU stay > 1 day (LOS2) (n = 29). Measurements were analyzed by groups using the Kruskal-Wallis and Mann-Whitney tests, and linear regression. MEASUREMENTS: Demographics, comorbidity, length of surgery, surgical procedure, and intraoperative fluids were recorded. MAIN RESULTS: The important differences in perioperative fluid management among the three groups included estimated blood loss (612 +/- 480 mL, 1853 +/- 1175 mL, 2702 +/- 1771 mL, means +/- SD); total crystalloid administration (2715 +/- 1396 mL, 5717 +/- 2574 mL, 7281 +/- 3417 mL); and total blood administration (92 +/- 279 mL, 935 +/- 757 mL, 1542 +/- 1230 mL) in LOS0, LOS1, and LOS2, respectively. The mixture of surgical procedures was similar in LOS1 and LOS2; and differed from LOS0. Predictors of ICU LOS included age, ASA physical status, surgical procedure, total crystalloid administration, and platelet administration. Surgical procedure and total crystalloid administration correlated (Pearson correlation coefficient = 0.441; p = 0.000) and were not related to age or ASA physical status. CONCLUSIONS: Total crystalloid administration during spine surgery does predict ICU LOS. In addition, total crystalloid administration is closely related to the surgical procedure. Given that the mixture of surgical procedures was similar in LOS1 and LOS2, but differed in estimated blood loss, total crystalloid administration, and total blood administration; intraoperative fluid management during spine surgery only predicts ICU LOS insofar as total crystalloid administration is related to the surgical procedure.

Postirradiation hyperthermia selectively potentiates the merocyanine 540-sensitized photoinactivation of small cell lung cancer cells.

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high-dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long-term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540-mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (< or = 42 degrees C, 3 h) potentiates the MC540-mediated photoinactivation of both wild-type (H69) and cisplatin-resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540-PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.

Soluble polymer-supported chemoenzymatic synthesis of the C(21)-C(27) fragment of the bryostatins.

A chemoenzymatic synthesis of the C(21)-C(27) fragment of the marine macrolide family of bryostatin antibiotics is presented. The approach commences from achiral starting materials and has as its crucial step the enzymatic resolution of a racemic mixture of soluble polymer-supported alcohols (syn-10 and syn-11). The immobilized lipase from Candida antarctica (Novozym 435) catalyzes the enantioselective acetylation of syn-10 (in 40% conversion and >99% ee), allowing isolation of the key intermediate (R)-14 in enantiomerically pure form following its cleavage from the poly(ethylene) glycol (PEG) scaffold. The PEG matrix is both compatible with the multipolymer enzymatic transformation and allows for rapid purification and facile NMR characterization of all intermediates throughout the synthesis.

Estrogen decreases infarct size after temporary focal ischemia in a genetic model of type 1 diabetes mellitus.

BACKGROUND AND PURPOSE: It is unclear how genetic type 1 diabetes mellitus (DM) influences infarct size when blood glucose is tightly controlled. The aim of this study was to determine the effect of genetic type 1 DM, as occurs in BB rats, on infarct size after transient unilateral middle cerebral artery occlusion (MCAO) in male and female rats. In addition, studies suggest that male type 1 DM rats have a higher incidence of end-organ complications than do females. A second aim of this study was to determine the effect of chronic 17beta-estradiol (E(2)) administration on infarct size in male BB rats. METHODS: Diabetic male (MDiab, n=14) and female (FDiab, n=8) BB rats were studied and compared with background strain Wistar rats (MWist, n=16; FWist, n=14). Two additional male cohorts (MWist+E(2), n=15; MDiab+E(2), n=14) received subcutaneous 25 microg E(2) implants 7 to 10 days before MCAO. Rats underwent 1 hour of MCAO followed by 22 hours of reperfusion. Physiological variables were controlled among groups, and the intraischemic laser Doppler flow signal was reduced similarly in all animals. Infarction volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining and image analysis. RESULTS: Preischemic blood glucose was 94+/-5, 127+/-13, 90+/-15, 63+/-18, 122+/-8, and 81+/-14 mg/dL in MWist, FWist, MDiab, FDiab, MWist+E(2), and MDiab+E(2) rats, respectively (mean+/-SE). Intraischemic laser Doppler flow was reduced to 20% to 25% of baseline in all groups. Striatal infarct size (percentage of ipsilateral caudate putamen) was increased in male diabetic rats relative to nondiabetic MWist rats (41+/-3% versus 28+/-3%). Striatal injury was not increased in FDiab rats, and infarction volume was smaller than that in FWist rats (23+/-4% in FWist versus 13+/-3% in FDiab). Chronic estrogen treatment reduced cortical and striatal infarction in MDiab+E(2) rats compared with untreated MDiab rats. CONCLUSIONS: Type 1 DM is associated with increased infarct size after temporary MCAO, despite tight control of blood glucose. The deleterious effect of DM is evident only in males rats; female diabetic BB rats sustain small infarcts. Chronic E(2) treatment reduced injury in the male BB rat, providing neuroprotection even in the presence of DM. These data suggest that genetic diabetes even with mild glucose elevation plays a role in determining neuropathology in experimental stroke. However, factors such as reproductive steroids also determine outcome in DM stroke.

Effect of molecular structure on the performance of triarylmethane dyes as therapeutic agents for photochemical purging of autologous bone marrow grafts from residual tumor cells.

Extensively conjugated cationic molecules with appropriate structural features naturally accumulate into the mitochondria of living cells, a phenomenon typically more prominent in tumor than in normal cells. Because a variety of tumor cells also retain pertinent cationic structures for longer periods of time compared with normal cells, mitochondrial targeting has been proposed as a selective therapeutic strategy of relevance for both chemotherapy and photochemotherapy of neoplastic diseases. Here we report that the triarylmethane dye crystal violet stains cell mitochondria with efficiency and selectivity, and is a promising candidate for photochemotherapy applications. Crystal violet exhibits pronounced phototoxicity toward L1210 leukemia cells but comparatively small toxic effects toward normal hematopoietic cells (murine granulocyte-macrophage progenitors, CFU-GM). On the basis of a comparative examination of chemical, photochemical, and phototoxic properties of crystal violet and other triarylmethane dyes, we have identified interdependencies between molecular structure, and selective phototoxicity toward tumor cells. These structure-activity relationships represent useful guidelines for the development of novel purging protocols to promote selective elimination of residual tumor cells from autologous bone marrow grafts with minimum toxicity to normal hematopoietic stem cells.

Apoptosis and necrosis occur in separate neuronal populations in hippocampus and cerebellum after ischemia and are associated with differential alterations in metabotropic glutamate receptor signaling pathways.

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1alpha, mGluR5, phospholipase Cbeta (PLCbeta), and Galphaq/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1alpha and mGluR5 were decreased but PLCbeta was increased. By immunocytochemistry, mGluR and PLCbeta immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.

Genetic variability in the response of normal murine hematopoietic progenitor cells to extracorporeal photochemotherapy.

Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 x 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.

Exploring the scope of poly(ethylene glycol) (PEG) as a soluble polymer matrix for the Stille cross-coupling reaction.

The optimization and efficient parallel synthesis and purification of a library of biaryl, heterobiaryl, and styryl derivatives, via the first reported poly(ethylene glycol)-supported palladium-catalyzed Stille procedure, are described. Preliminary investigations into the reaction between monomethoxy poly(ethylene glycol)5000-supported iodide 1a with tributylphenyltin 2 revealed that the optimal "liquid-phase" conditions employ PdCl2(PPh3)2 (0.1 equiv) catalysis with LiCl (10 equiv) in DMF at 80 degrees C for either 48 h (at 20 mM concentration of 1a) or 24 h (at 10 mM concentration of 1a). The soluble polymer-supported reaction is superior to its solution-phase counterpart because the tributyltin side products and excess reagents are easily separated from the product intermediate 3a by precipitation of 3a into diethyl ether followed by recovery of the polymer by filtration in > 99%. In addition, the homocoupled byproduct 6 is also removed during this precipitation step. Under these conditions the transesterified biaryl adduct 4a can be isolated in 97-98% yield. The scope of this reaction was probed in a parallel format with the PEG-supported electrophiles 1a-b and a range of tributyl stannanes 2 and 7-13 under the optimized conditions vide supra. Subsequent cleavage of the polymer-supported adducts, by transesterification, and short column chromatography yielded a library of substituted methyl benzoates 4a-b and 14a-b to 20a-b in high yield (69-99%) and purity (> 95%).

Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis.

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimer's disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-D-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis-necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.

Protein kinase C expression and activity after global incomplete cerebral ischemia in dogs.

BACKGROUND AND PURPOSE: Studies suggest that protein kinase C (PKC) activation during ischemia plays an important role in glutamate neurotoxicity and that PKC inhibition may be neuroprotective. We tested the hypothesis that elevations in the biochemical activity and protein expression of Ca2+-dependent PKC isoforms occur in hippocampus and cerebellum during the period of delayed neurodegeneration after mild brain ischemia. METHODS: We used a dog model of 20 minutes of global incomplete ischemia followed by either 6 hours, 1 day, or 7 days of recovery. Changes in PKC expression (Western blotting and immunocytochemistry) and biochemical activity were compared with neuropathology (percent ischemically damaged neurons) by means of hematoxylin and eosin staining. RESULTS: The percentage of ischemically damaged neurons increased from 13+/-4% to 52+/-10% in CA1 and 24+/-11% to 69+/-6% in cerebellar Purkinje cells from 1 to 7 days, respectively. The occurrence of neuronal injury was accompanied by sustained increases in PKC activity (240% and 211% of control in hippocampus and cerebellum, respectively) and increased protein phosphorylation as detected by proteins containing phosphoserine residues. By Western blotting, the membrane-enriched fraction showed postischemic changes in protein expression with increases of 146+/-64% of control in hippocampal PKCalpha and increases of 138+/-38% of control in cerebellar PKCalpha, but no changes in PKCbeta and PKCgamma were observed. By immunocytochemistry, the neuropil of CA1 and CA4 in hippocampus and the radial glia in the molecular layer of cerebellum showed increased PKCalpha expression after ischemia. CONCLUSIONS: This study shows that during the period of progressive ischemic neurodegeneration there are regionally specific increases in PKC activity, isoform-specific increases in membrane-associated PKC, and elevated protein phosphorylation at serine sites.

Photochemical purging of autologous bone marrow grafts: assessment of damage to stem cells and the microenvironment in long-term marrow cultures.

Toxicity of merocyanine 540 (MC540) was assessed in long-term (Dexter-type) bone marrow cultures and in a short-term in vitro clonal assay of fibroblast colony-forming cells (CFU-F). Exposure of freshly explanted mouse bone marrow cells to MC540 (15 micrograms/ml) and white light (fluence: 126 kJ/m2) reduced CFU-F by approximately 2 logs but did not abrogate the cells' capacity to establish and maintain long-term bone marrow cultures. Fat cells were rare or absence in adherent layers established with photosensitized bone marrow cells but the cultures' capacity to generate non-adherent cells, granulocyte/macrophage progenitors (CFU-GM), and early erythroid progenitors (BFU-E) was only moderately (28-36%) reduced.

Role of cytoprotective mechanisms in the photochemical purging of autologous bone marrow grafts.

The molecular basis of the differential sensitivity of normal hematopoietic stem cells and of leukemia, lymphoma, and neuroblastoma cells to merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) is not yet completely understood. While the capacity to bind dye molecules appears to be the major determinant of a cell's susceptibility of MC540-mediated PDT, we here present evidence that under certain experimental conditions a cell's capacity to repair MC540-mediated photodynamic damage is also an important factor. Two parameters, temperature and intracellular glutathione (GSH) content, were varied to investigate the role of cellular defense mechanisms in the dye-sensitized photoinactivation of normal murine granulocyte/macrophage progenitors (CFU-GM) and K562, L1210, and melphalan-resistant L1210/L-PAM1 leukemia cells. When exposed to MC540 and light at room temperature, the three leukemia cell lines bound similar amounts of dye and accumulated similar amounts of lipid hydroperoxide (LOOH) but differed markedly in their sensitivity to MC540-mediated PDT. Performing MC540-mediated PDT at 4 degrees C instead of at room temperature reduced dye binding and LOOH generation and enhanced cytotoxicity in some but not all cell lines. A brief (< or = 120 minutes) incubation at 37 degrees C immediately following MC540-mediated PDT accelerated the decay of LOOH in all leukemic cell lines and reduced cell kill by about 2 log in both CFU-GM and leukemia cells. The effect of post-PDT incubation at 37 degrees C on LOOH decay was most pronounced in K562 and least pronounced in L1210/L-PAM1 cells, whereas its effect on cell survival was less pronounced in L1210 cells than in the remaining cell types. L1210/L-PAM1 cells whose GSH content had been reduced from 8.2 to 1.6 micrograms/mg protein by incubation with buthionine sulfoximine recovered from potentially lethal photodynamic damage as rapidly as untreated L1210/L-PAM1 cells and more rapidly than wild-type L1210 cells with a GSH content of 4.5 micrograms/mg protein. Thus, with regard to capacity of L1210/L-PAM1 cells to recover from photodynamic damage, the cells' enhanced capacity to synthesize GSH appeared more decisive than intracellular GSH levels per se. Taken together, these data suggest that temperature-dependent cellular defense mechanisms are significant determinants of a cell's susceptibility to MC540-mediated PDT. The data emphasize the need for temperature control during and immediately after the photochemical purging of autologous bone marrow or peripheral blood stem cells.