Brain Functional Changes before, during, and after Clinical Pain.

This study used an emerging brain imaging technique, functional near-infrared spectroscopy (fNIRS), to investigate functional brain activation and connectivity that modulates sometimes traumatic pain experience in a clinical setting. Hemodynamic responses were recorded at bilateral somatosensory (S1) and prefrontal cortices (PFCs) from 12 patients with dentin hypersensitivity in a dental chair before, during, and after clinical pain. Clinical dental pain was triggered with 20 consecutive descending cold stimulations (32° to 0°C) to the affected teeth. We used a partial least squares path modeling framework to link patients' clinical pain experience with recorded hemodynamic responses at sequential stages and baseline resting-state functional connectivity (RSFC). Hemodynamic responses at PFC/S1 were sequentially elicited by expectation, cold detection, and pain perception at a high-level coefficient (coefficients: 0.92, 0.98, and 0.99, P < 0.05). We found that the pain ratings were positively affected only at a moderate level of coefficients by such sequence of functional activation (coefficient: 0.52, P < 0.05) and the baseline PFC-S1 RSFC (coefficient: 0.59, P < 0.05). Furthermore, when the dental pain had finally subsided, the PFC increased its functional connection with the affected S1 orofacial region contralateral to the pain stimulus and, in contrast, decreased with the ipsilateral homuncular S1 regions ( P < 0.05). Our study indicated for the first time that patients' clinical pain experience in the dental chair can be predicted concomitantly by their baseline functional connectivity between S1 and PFC, as well as their sequence of ongoing hemodynamic responses. In addition, this linked cascade of events had immediate after-effects on the patients' brain connectivity, even when clinical pain had already ceased. Our findings offer a better understating of the ongoing impact of affective and sensory experience in the brain before, during, and after clinical dental pain.

Place cell rigidity correlates with impaired spatial learning in aged rats.

In humans and in animals, some aged individuals are severely impaired in learning and memory capacity whereas others perform as well as young adults. In the present study, the spatial memory capacity of young and aged rats was characterized by the Morris water maze task, and then firing patterns of hippocampal "place cells" were assessed as the animals explored a familiar environment and a geometrically-altered version of the environment. Spatial representations of hippocampal cells in young and memory-intact aged rats changed upon exposure to the altered environment. In contrast, spatial representations of many cells in aged, memory-impaired rats were unaffected by the environmental alteration. Furthermore, combining all groups, the extent to which spatial representations distinguished the familiar and altered environments predicted learning capacity in the water maze. These findings suggest that a major component of memory impairment in aging may be the failure of the hippocampus to encode subtle differences in contextual information that differ across multiple experiences, such as the sequence of training trials in the water maze.

Rat RT1.B-transfected fibroblast lines process and present myelin antigens and activate T cells to induce experimental autoimmune encephalomyelitis.

The genes encoding the Lewis rat RT1.B molecule (MHC Class II I-A equivalent) were transfected and expressed in mouse DAP.3 fibroblast cells together with the gene encoding the mouse ICAM-1 molecule. Both molecules were stably expressed on the cell surface of DAP.3 cells under longterm culture conditions. The RT1.B/mICAM-1 transfectants presented antigen in a specific manner to a RT1. B-restricted rat T cell hybridoma specific for the 69-89 peptide of myelin basic protein (BP). In addition, the transfectants were able to present antigen to a BP69-89-specific rat T cell line. Presentation to a RT1.D (MHC Class II I-E equivalent)-restricted BP87-99-specific T cell line was minimal. Production of the Th1 cytokine IFN-gamma by BP69-89-specific T cells when stimulated by RT1.B/mICAM-1 transfectants correlated very well with proliferation to specific antigen. Moreover, RT1.B-transfected DAP.3 cells sufficiently stimulated BP69-89-specific T cells such that they were able to transfer experimental autoimmune encephalomyelitis (EAE) to Lewis rat recipients. Thus, the RT1.B molecule is functionally expressed on the surface of transfected Dap.3 fibroblasts and is capable of MHC Class II-restricted, antigen-specific presentation to rat T cells.

Orbitofrontal cortex and representation of incentive value in associative learning.

Clinical evidence indicates that damage to ventromedial prefrontal cortex disrupts goal-directed actions that are guided by motivational and emotional factors. As a consequence, patients with such damage characteristically engage in maladaptive behaviors. Other research has shown that neurons in the corresponding orbital region of prefrontal cortex in laboratory animals encode information regarding the incentive properties of goals or expected events. The present study investigates the effect of neurotoxic orbitofrontal cortex (OFC) lesions in the rat on responses that are normally influenced by associations between a conditioned stimulus (CS) and the incentive value of reinforcement. Rats were first trained to associate a visual CS with delivery of food pellets to a food cup. As a consequence of learning, rats approached the food cup during the CS in anticipation of reinforcement. In a second training phase, injection of LiCl followed consumption of the food unconditioned stimulus (US) in the home cage, a procedure used to alter the incentive value of the US. Subsequently, rats were returned to the conditioning chamber, and their responding to the CS in the absence of the food US was tested. Lesions of OFC did not affect either the initial acquisition of a conditioned response to the light CS in the first training phase or taste aversion learning in the second training phase. In the test for devaluation, however, OFC rats exhibited no change in conditioned responding to the visual CS. This outcome contrasts with the behavior of control rats; after devaluation of the US a significant decrease occurred in approach to the food cup during presentation of the CS. The results reveal an inability of a cue to access representational information about the incentive value of associated reinforcement after OFC damage.

Apoptosis and the cell cycle in Xenopus laevis: PHA and PMA exposure of splenocytes.

T cell receptor (TCR) ligation and protein kinase C (PKC) activation stimulate proliferation and modulate apoptosis in both mammalian and amphibian lymphocytes. The potential relationship between apoptosis and the cell cycle in mature Xenopus laevis splenic lymphocytes is addressed by monitoring apoptosis and DNA synthesis over time, using incorporation of propidium iodide (PI) and flow cytometry. Aliquots of the same populations of cells are followed after exposure in vitro to phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA). Significant increases in apoptosis preceed those in DNA synthesis by 12 to 16 h following exposure to both reagents. Since apoptosis preceeds DNA synthesis, these dying cells clearly do not need to enter the S phase of the cell cycle before becoming apoptotic, in contrast to mammalian T cells. Another striking difference is that the reagent with weaker mitogenic properties in this species, PHA, is significantly a more potent apoptogen, than the strong mitogen, PMA. The two phenomena then appear to be inversely related in Xenopus cells. Data on DNA synthesis suggest independence of the two phenomena, as DNA synthesis is stimulated in direct proportion to the strength of each reagent as a mitogen. Mature mammalian T-cells undergo apoptosis only when previously activated. The Xenopus lymphocytes examined were not deliberately activated by exposure to antigen or lectin. PMA, a cancer promoter in mammals, usually 'rescues' mammalian cells from apoptosis, but stimulates apoptotic increases in Xenopus cells. Thus, mature Xenopus lymphocytes may be more readily stimulated to die by cancer inducing agents than mammalian lymphocytes. This could make them less susceptible to transformation into immortalized cancer cells. This characteristic may considerably contribute to the observed resistance to spontaneous and chemically-induced neoplasia in wild type, non-isogeneic or non-inbred Xenopus.

Expression of a Fas-like proapoptotic molecule on the lymphocytes of Xenopus laevis.

Ligation of the externally expressed Fas (APO1/CD95) molecule will initiate programmed cell death (apoptosis), in many mammalian developing and adult cells. Fas-induced apoptosis has not been demonstrated with the cells of any non-mammalian vertebrate. We immunostained suspensions of splenocytes from adult Xenopus laevis, the South African clawed toad, with a polyclonal rabbit anti-human Fas antibody raised against the amino acid residues 321-335 of human Fas. The binding was specific, as it was dramatically reduced by preincubation of the antibody with the Fas peptide used to make it, but not with a Fas-ligand (FasL) peptide. The binding was enhanced after in vitro exposure of the splenocytes to phytahemagglutinin (PHA), a T cell mitogen and apoptogen in this species. Sections of developing Xenopus larval tissue were also immunostained with the polyclonal rabbit anti-human Fas antibody. Consistent binding of thymocytes and splenocytes was not observed until early metamorphosis in these immunological sites. A monoclonal mouse anti-human Fas antibody, previously used to stimulate apoptosis in mammalian cells, induced significant levels of apoptosis in adult Xenopus splenocytes and additionally, bound specifically to a splenocyte extract, as assayed by ELISA. Thus, a molecule on Xenopus splenocytes shares both structural and functional homologies with human Fas, indicating the evolutionary conservation within vertebrates of this means of initiating apoptosis.

A re-examination of the role of basal forebrain cholinergic neurons in spatial working memory.

The basal forebrain cholinergic system, which innervates widespread cortical and limbic structures, has traditionally been considered important for learning and memory. The use of an immunotoxin, 192 IgG-saporin, has brought this functional designation into question; selective immunolesions of basal forebrain cholinergic neurons have failed to reproduce a number of behavioral deficits that were observed with less selective lesion methods. Recent reports, however, have indicated that a mild impairment is observed in rats on a spatial working memory task after 192 IgG-saporin lesions of the rostral groups of cholinergic neurons located in the medial septal area (MSA). Those studies used a lesion protocol in which a single large volume injection of the immunotoxin was made into the MSA. In the current study, multiple small injections were made at the locations of cholinergic neurons in the MSA, producing a cholinergic depletion comparable to that reported in the earlier studies where deficits were observed. In the current study, however, rats with cholinergic lesions had no impairment in the spatial working memory task, even when delays ranging from 60 s to 8 h were imposed within a trial. The current report indicates that selective removal of cholinergic neurons in the basal forebrain may not be sufficient to produce a deficit in spatial working memory.

The role of an amygdalo-nigrostriatal pathway in associative learning.

The present study examined the role of an amygdalo-nigrostriatal pathway in associative learning. An asymmetrical lesion model was used to test whether a circuit from the amygdala central nucleus to the dorsolateral striatum, via the substantia nigra, is critical for mediating conditioned orienting responses. Rats with an asymmetrical lesion, consisting of neurotoxic removal of central nucleus neurons in one hemisphere and depletion of the dopamine innervation of the dorsolateral striatum in the contralateral hemisphere, failed to acquire conditioned orienting responses. In contrast, the asymmetrical lesion had no effect on spontaneous orienting or learning another response directed to the source of the food unconditioned stimulus in the same task. A second experiment tested the effect of reversible inactivation of the dorsolateral striatum contralateral to a neurotoxic central nucleus lesion on acquisition of the conditioned orienting response. Although inactivation did not affect spontaneous orienting, rats failed to acquire the conditioned orienting response during sessions in which inactivation occurred. Immediately after the inactivation procedure was terminated, however, a significant increase in orienting to the conditioned stimulus was evident. These data support the interpretation that the dorsolateral striatum provides a route for the expression of the conditioned orienting response but is not essential for acquisition of this learned behavior.

Spatial learning and memory at defined points of the estrous cycle: effects on performance of a hippocampal-dependent task.

Learning based on hippocampal-dependent spatial navigation in female rats was assessed at identified points in the estrous cycle corresponding to low (estrus) and high (proestrus) circulating estrogen. With background training in water-maze procedures, rats learned the location of an escape platform in the maze in a single session of 8 training trials. A strong spatial bias for the escape platform was also evident in a probe trial used to assess retention of learning 30 min after the training session. This entire protocol was completed in less than an hour. The performance of the estrus and proestrus rats was indistinguishable on all behavioral measures, irrespective of the stage of estrous cycle during the task. These results indicate that rapid learning and retention for spatial information over a relatively short interval may be preserved despite morphological alterations in hippocampal dendritic spine density in the normally cycling female rat.

Selective immunolesions of hippocampal cholinergic input fail to impair spatial working memory.

The septo-hippocampal cholinergic pathway has traditionally been thought of as essential for spatial memory. Recent studies have demonstrated intact spatial learning following removal of this pathway with an immunotoxin selective for cholinergic neurons. In the present experiment, rats with selective removal of hippocampal cholinergic input were tested in a delayed nonmatching-to-position task in a water version of the radial arm maze. This allowed us to increase and parametrically vary the memory load compared with the standard Morris water maze (by varying the delay between the initial four choices and the final four choices) to determine if this would reveal a deficit in rats with lesions of septo-hippocampal cholinergic projections. Male Long-Evans rats were given injections of 192 IgG-saporin, a selective immunotoxin for cholinergic neurons, into the medial septum/vertical limb of the diagonal band (MS/VDB) to remove cholinergic projections to the hippocampus, or a control surgery. The rats were trained on the radial maze task following surgery. An escape platform was located at the end of each arm of the maze and was removed after an arm was utilized for escape. After initial training, a delay was interposed between the first four trials and the second four trials. Errors during the second four-trial component were scored in two categories: retroactive (reentering an arm chosen before the delay) and proactive (reentering an arm chosen after the delay). Retroactive errors increased as delay increased (from 60 s to 6 h) but were equivalent in control and MS/VDB-lesion groups. Proactive errors did not vary with delay and were also unaffected by the lesion. Radioenzymatic assays for choline acetyltransferase activity in the hippocampus of lesioned rats confirmed a significant loss of cholinergic input from the MS/VDB. These results indicate that normal spatial working memory is possible after substantial loss of septo-hippocampal cholinergic projections.

Prospective comparative trial of short course (four day) and continuous tobramycin in combination with cefoperazone or mezlocillin in febrile, granulocytopenic patients.

In a prospective, randomized trial of 195 febrile episodes in granulocytopenic patients short course aminoglycoside treatment (initial tobramycin and cefoperazone followed by tobramycin discontinuation at day four of therapy) was compared with two regimens (tobramycin plus cefoperazone and tobramycin plus mezlocillin) in which both drugs were continued for up to 26 days. All regimens were successful as empirical therapy with comparable response rates of just over seventy per cent. Fifty-three per cent of the initial episodes of fever were related to documented infections which responded less well (P = 0.007) than unexplained fever. Patients with bacteraemia, pneumonia or Gram-positive aerobic or Pseudomonas aeruginosa infections responded poorly to all regimens. The recovery from granulocytopenia was the most important determinant of successful response. Aminoglycoside discontinuation followed by cefoperazone monotherapy after day four was statistically as effective as the combination regimens. Short course tobramycin therapy eliminated the nephrotoxicity seen in the combination limbs. The use of cefoperazone was not associated with an increased incidence of hypoprothrombinemia; however, the only three bleeding episodes occurred in patients given cefoperazone but not vitamin K. Short course aminoglycoside therapy will reduce cost and nephrotoxicity when compared with prolonged combination therapy and should be further explored in this setting, with use of different agents and comparison with monotherapy.

Moderate-dose vincristine infusion in refractory breast cancer.

Continuous infusion of vincristine at the maximally tolerated infusion dosage of 0.5 mg/m2/day for 5 days has been investigated in 15 patients with refractory breast cancer. Infusion courses were repeated every 3 weeks in the absence of disease progression or prohibitive toxicity. Progressive disease was observed in 14 patients. A partial response lasting 2 months occurred in a patient with pulmonary and skin metastases who had previously received vincristine by bolus injection. Toxicity consisted primarily of mild neurotoxicity of similar degree to that expected with bolus injection. Thrombocytopenia occurred, but was uncommon. The cumulative response rate at this dosage level (2/16, 13%) in our phase I-II trials indicates very limited clinical activity of vincristine infusion in advanced, refractory metastatic breast cancer.

Clinical trial of folinic acid to reduce vincristine neurotoxicity.

In a murine model system, folinic acid demonstrated host-protective properties during administration of repetitive and lethal doses of vincristine (VCR). Subsequently, folinic acid was evaluated in patients receiving VCR during an adjuvant chemotherapy program for stage II carcinoma of the breast. The toxicities, cumulative VCR dosage, and percentage of ideal dosage observed in 18 patients receiving folinic acid have been compared with those observed in 70 patients who previously received VCR without folinic acid in the same chemotherapy program. All patients ideally were intended to receive VCR 1.0 mg/m2 weekly for 6 weeks, with dose modification for neurotoxicity. Treatment patients received folinic acid 800 mg PO daily in three divided doses during the 6-week course. The degree of neurotoxic manifestations of VCR was similar in the treatment and comparison patients. Absent to mild neurotoxicity was observed in approximately 70% of patients in both groups; moderate or greater neurotoxicity occurred in about 30% of patients in both groups. Full dosage (6.0 mg/m2) was attained in 7 (39%) treatment patients and 17 (24%) comparison patients (P = 0.21). The mean percentage of the ideal dosage of VCR was 73.7 +/- 28.7 in patients receiving folinic acid and 76.1 +/- 20.5 in those given only VCR (P = 0.69). Hematologic toxicities were similar in both groups, but nausea occurred more frequently in the folinic acid group. Folinic acid in this dose and schedule afforded no protection from the neurotoxic side effects of VCR.

Clinical trial of pyridoxine to reduce vincristine neurotoxicity.

In a murine model system, pyridoxine has demonstrated protective properties during administration of lethal doses of vincristine (VCR). Subsequently, pyridoxine has been evaluated in patients receiving VCR during an adjuvant chemotherapy program for stage II carcinoma of the breast. The toxicities, cumulative VCR dosage, and percentage of ideal dosage observed in 24 patients receiving pyridoxine have been compared to those observed in 88 patients who previously received VCR without pyridoxine in the same chemotherapy program. All patients ideally were to receive VCR 1.0 mg/m2 weekly for 6-weeks with dose modification for neurotoxicity. Treatment patients received pyridoxine 1.5 grams p.o. daily in three divided doses during the 6-week course. The degree of neurotoxic manifestations of VCR was similar in the treatment and comparison patients. Absent to mild neurotoxicity was observed in approximately 70% of patients in both groups; moderate or greater neurotoxicity occurred in about 30% of patients in both groups. Full dosage (6.0 mg/m2) was attained in 8 (33%) treatment patients and 18 (24%) comparison patients (p = 0.28). The mean percentage of ideal dosage of VCR was 84.6 +/- 10.8 in patients receiving pyridoxine and 81.9 +/- 21.6 in those given only VCR (p = 0.59). Gastrointestinal and hematologic toxicities were similar in both groups. Pyridoxine in this dose and schedule afforded no protection from the neurotoxic side effects of VCR.

Sequential hemibody and local irradiation with combination chemotherapy for small cell lung carcinoma: a preliminary analysis.

Sequential hemibody irradiation (SHB) was integrated with combination chemotherapy and local irradiation (LRT) in the induction and consolidation phases of a therapeutic protocol for small cell lung carcinoma (SCLC). Forty-one previously untreated patients were entered into this program. Among 38 evaluable patients (20 with limited disease [LD] and 18 with extensive disease [ED], the overall response rate was 63% (90% in LD and 33% in ED patients). The estimated overall survival is 8.1 months. The major toxicity has been myelosuppression--especially thrombocytopenia. The frequency of previously described "acute radiation syndromes" and radiation pneumonitis associated with hemibody irradiation have been substantially decreased at the current dosage with premedication and shielding techniques. The integration of SHB as a systemic therapy with combination chemotherapy and LRT is a feasible program for sequential administration of non-cross-resistant agents in SCLC and may be beneficial in patients with limited disease.