Performance of a novel BD stem cell enumeration kit on two flow cytometry systems.

INTRODUCTION: Hematopoietic stem cell transplantation, which requires accurate enumeration of stem cells, is routinely used in clinical settings. Flow cytometry provides a qualitative and quantitative assessment of CD34⁺ cells. Precision, linearity, and stability of the novel BD™ Stem Cell Enumeration (SCE) Kit were evaluated on two flow cytometry platforms using a modified ISHAGE gating strategy and including a viability dye for data acquisition and analysis. METHODS: Precision and linearity were evaluated on BD FACSCanto™ II and BD FACSCalibur™ systems. Stability was evaluated on the BD FACSCanto II system. Precision was tested using both high and low controls. Linearity was evaluated using dilutions from CD34⁺ cell pools, while stability was evaluated using fresh leukapheresis specimens. RESULTS: Both systems showed precision with limited variability in absolute counts and percentages of viable CD34⁺ cells. The linearity range of viable CD34⁺ cells in both systems was established at 0-1000 cells/µL, showing a linear relationship (R² = 0.99). Stability of CD34⁺ cells in mobilized leukapheresis samples was confirmed up to 24 h after collection and up to 60 min after the end of stain/lyse procedures. CONCLUSION: The BD SCE Kit on both flow cytometry systems shows consistent and reproducible results.

A stable prostacyclin analog enhances ectopic activity in rat sensory neurons following neuropathic injury.

Prostanoids sensitize sensory afferents during inflammation. However, their role in neuropathic pain is still unclear. We analyzed the actions of prostanoids, non-selective (indomethacin) or selective (celecoxib and NS-398) cyclooxygenase-2 (COX or COX-2) inhibitors, on the ectopic activity of dorsal root ganglia (DRG) and dorsal horn (DH) neurons in a model of neuropathic injury. Extracellular recordings of DRG and DH neurons and cardiovascular measurements were performed on anesthetized, paralyzed and artificially ventilated adult male Sprague-Dawley rats whose sciatic nerve had been transected. PGD(2), PGE(2), PGF(2alpha), carbaprostacyclin (cPGI(2); a stable prostacyclin analog), and carbocyclic thromboxane (cTXA(2)) were administered at cumulative doses (0.0001-5 mg/kg, i.p.) at 5 or 10 min intervals. Only cPGI(2) significantly increased the DRG and DH activity in a dose-dependent manner, with ED(50) values of 0.05 (0.01-0.96) and 0.69 (0.11-1.04) mg/kg, respectively. The other prostanoids did not significantly increase activity, although they reduced heart rate for up to 5 min following administration. Time course experiments with single doses of cPGI(2) (1 mg/kg, i.v.) increased DH discharge rate 3-17 min after injection. Indomethacin (3 mg/kg, s.c.), but not celecoxib or NS-398 (both at 6 mg/kg, s.c.), reduced both DRG and DH activity. Our results indicate that cPGI(2) excites DRG and DH neurons of neuropathic rats, and may suggest a role for IP prostanoid receptors in pain episodes associated with nerve injury. The inhibitory effect of indomethacin, but not celecoxib or NS-398, on ectopic activity may suggest that a tonic generation of PGI(2) by COX-1 could contribute to neuropathic pain.

Opioid peptide systems following a subconvulsant dose of pentylenetetrazol in rats.

The development of epilepsy and a progressive increase in susceptibility to seizures may involve changes in inhibitory and excitatory systems from the beginning of the process. The present study was focused to analyze the opioid peptide changes induced by a chemical sub-convulsant stimulation. Experiments were carried out to determine opioid peptide release, mu receptor binding and proenkephalin expression in rat brain, as well as nociceptive responses, following the administration of a sub-convulsant dose of pentylenetetrazol (PTZ) (30 mg/kg, i.p.). Membrane binding experiments revealed reduced number of mu binding sites (Bmax) in cortex and amygdala, but not in striatum and hippocampus, an effect that was evident only 24 h, but not 28 days, after PTZ treatment. In situ hybridization experiments suggested a significant enhancement of proenkephalin mRNA expression in specific brain regions 24 h after PTZ treatment. Microdialysis combined with a universal opioid peptide radioimmunoassay revealed extracellular opioid peptide levels to be elevated in the amygdala (137%) 90 min after PTZ administration. Evaluation of nociceptive responses using the Randall-Selitto test showed an analgesic effect short term (30-90 min) after PTZ injection. Collectively, these data provide evidence for a significant activation of opioid peptide systems as a consequence of the administration of a sub-convulsant dose of PTZ. These neurochemical changes may play an important role in the progression of epileptogenesis.

QX-314 inhibits ectopic nerve activity associated with neuropathic pain.

In animal models of neuropathic pain, transection or constrictive injury to peripheral nerves produces ectopic discharges originating at both injury sites and related dorsal root ganglia (DRG). In addition, hyperexcitability is observed in associated dorsal horn (DH) neurons of the spinal cord. As ectopic discharges are inhibited by agents that block voltage-sensitive Na+ channels, it has been postulated that accumulation of Na+ channels in the membrane at nerve injury sites may contribute to the development of ectopic nerve activity (ENA). The goal of the present study was to compare the sensitivity of ENA to lidocaine and QX-314, a positively charged lidocaine derivative, which is frequently assumed to be membrane impermeant. Experiments were performed on adult male Sprague-Dawley rats in which the common sciatic nerve had been transected 4-10 days earlier. Extracellular microelectrode recordings were made from DRG and DH neurons, and neuronal activity was measured in fine bundles of microfilaments teased from sciatic nerves in anesthetized and paralyzed rats. Comparative effects on heart rate (HR) and mean blood pressure (MBP) were also studied. To confirm that externally applied QX-314 is able to inhibit high frequency activity in sensory nerves, QX-314 was superfused over isolated rat vagus nerves during stimulation of compound action potentials in C-fibers (C-spikes). As expected, intravenously administered lidocaine inhibited ENA at all three sites. Lidocaine ED50 values (expressed as mg/kg, with 95% confidence limits) were: 10.2 (7.8-13.3), 1.4 (0.8-2.4) and 0.9 (0.4-2.0) for neuromas, DRG and DH neurons, respectively. QX-314 also induced dose-dependent inhibition of ENA at neuromas and DRG, but produced only a small inhibition of DH neuron ENA. QX-314 had the following ED50 values (mg/kg) for neuromas, DRG and DH neurons, respectively: 2.3 (2.0-2.8), 6.9 (4.7-26.5) and 85.7. QX-314-mediated inhibition of DRG ENA had a slow onset and was long-lasting, relative to lidocaine. Lidocaine or QX-314 also significantly reduced HR and MBP in the same dose range as that which reduced ENA in DRG or neuromas. In isolated rat vagus nerve recordings, QX-314 induced marked use-dependent inhibition of C-spike amplitude, with IC50 values (microM) of 9000 (4600-18,000) and 350 (290-420) for low- (0.03 Hz) and high-frequency (30 Hz) C-spikes, respectively. These data support the hypothesis that Na+ channel accumulation contributes to the generation of ectopic discharges in neuromas and DRG, and suggests that intravenous QX-314 can acutely block Na+ channels at these sites.

Tetrodotoxin inhibits neuropathic ectopic activity in neuromas, dorsal root ganglia and dorsal horn neurons.

Neuropathic pain or persistent dysesthesias may be initiated by mechanical, chemical, or ischemic damage to peripheral sensory nerves. In animal models of neuropathic pain, transection or constrictive injury to peripheral nerves produces ectopic discharges originating at both injury sites and related dorsal root ganglia (DRG), and, consequently, hyperexcitability in associated dorsal horn (DH) neurons of the spinal cord. Since ectopic discharges are inhibited by agents that block voltage-sensitive Na+ channels, it has been postulated that accumulation of Na+ channels in the membrane at nerve injury sites may contribute to, or be responsible for, the development of ectopic neuronal activity (ENA). The present study therefore, tested the sensitivity of ENA to intravenously administered tetrodotoxin (TTX), an extremely potent and selective Na+ channel blocker. Comparative effects of TTX on cardiac parameters such as heart rate (HR) and diastolic blood pressure (DBP) were also studied. Experiments were performed on adult male Sprague-Dawley rats in which the common sciatic nerve had been transected 4-10 days earlier. Neuromal activity was measured in fine bundles of microfilaments teased from sciatic nerves, and extracellular microelectrode recordings were made from DRG and DH neurons. Cardiovascular parameters were recorded simultaneously. Intravenously administered TTX induced dose-dependent inhibition of ENA, with that originating from neuromas being the most sensitive; ED50 values (expressed as microg/kg, with 95% confidence limits) for neuromal, DRG and DH neuron activity were: 0.8 (0.6-1.2), 4.3 (2.2-8.4) and 36.2 (16.1-81.3), respectively. Inhibition of ENA in neuromas and DRG did not recover within 10 min after 100 or 300 microg/kg TTX. By comparison, the ED50 value for the initial decrease of HR was 17.9 (15.0-21.5) microg/kg, and partial recovery occurred within approximately 3 min. These data support the hypothesis that Na+ channel accumulation contributes to the generation of ectopic discharges in neuromas and DRG, and suggest that TTX-sensitive Na+ channels located at the nerve injury site and DRG play an important role in the genesis of neuropathic pain.

Cortical facilitatory action on centralis lateralis thalamic activity during the development of carrageenin-produced inflammation.

In order to understand the neuronal mechanisms involved in acute and chronic pain, we studied the thalamic and cortical control action, which allows the suppression of the neuronal responses to noxious stimulation. As an experimental pain model we used carrageenin injected in the paw of male Wistar rats. The tonic facilitatory cortical control on centralis lateralis thalamic nuclei (CL) activity is described at different times after carrageenin-produced inflammation. Simultaneous extracellular unit recordings were carried out at CL and medial prefrontal cortex (PCx) cells in anesthetized male Wistar rats. The PCx control was tested by blocking in a transient and reversible manner, using the cortical spreading depression (CSD). Carrageenin injection (1%; 0.2 ml) into the plantar surface of the right hind paw, and the influence of Lidocaine (2%; 0.2 ml) applied in the inflamed paw, was tested on unit activity in PCx and CL cells. Thalamic cells recorded in acute and subacute stages (24-72 h after carrageenin administration) were activated by tactile, light pressure and joint movement stimulation yielded before the injection. After carrageenin, the thalamic cells displayed spontaneous high frequency burst discharges, also presenting a progressive and significant increase (p < 0.001, ANOVA test) of their spontaneous firing rate when compared with control cell activity. Lidocaine reduced the enhanced activity induced by carrageenin in thalamic neurones (p < 0.001, Student t test). In PCx neurones were also recorded in acute and subacute stages. Cortical cells from acute and subacute group were activated by nociceptive and non-nociceptive stimulation. In acute stage, cortical cells increased their firing rate after carrageenin and we could not observe modifications upon their firing rate due to Lidocaine. The CSD blocked all cortical activity in acute and subacute stages. During the CSDs, overall thalamic activity was suppressed in neurones from acute (91%) and subacute (87%) stages. The blockage was observed when the propagated wave produced by CSD arrived into the medial prefrontal cortex. The CSD also suppressed the PCx and the CL noxious responses evoked by pressure in the receptive field. This study shows the tonic facilitatory control of the PCx upon intralaminar thalamic noxious responses, during acute and subacute stages of carrageenin produced-inflammation. In the literature, it has been proposed that the CL thalamic nuclei and the prefrontal cortex are involved in processing the affective component of pain. It may be possible to suppress the thalamic activity during chronic pain, using the transient and reversible blockage of CSD, giving rise to a reduction in the affective reactions to pain. This could also be a therapeutic alternative in chronic pain treatment.

Actions of sciatic nerve ligature on sexual behavior of sexually experienced and inexperienced male rats: effects of frontal pole decortication.

The action of the hyperalgesia produced by the loose ligature of the sciatic nerve on the sexual behavior of two groups of sexually experienced and inexperienced male rats was studied. The putative changes in the behavior induced by this manipulation were attempted to be counteracted by the removal of the anterior frontal cortex. Independently of the sexual experience, the ligature of the sciatic nerve did not modify any parameter of the sexual behavior, except for a slight prolongation of the intromission latency. Surprisingly, the removal of the cortical frontal pole resulted in drastic changes in the sexual behavior of sexually inexperienced male rats such as an increase in the intromission and ejaculation latencies and the postejaculatory interval. These changes were not observed in the sexually experienced group. Interestingly, the sciatic nerve ligature prevented the changes in the copulatory behavior produced by the frontal cortex removal. These results suggest that the hyperalgesia induced by the sciatic nerve ligature was unable to alter the sexual behavior. By contrast, the frontal cortex removal produced important changes in the sexual behavior that depend upon the previous sexual experience. All results are discussed on the bases of the neural control underlying the possible relationships between algesia and male sexual behavior.

Facilitating action of medial prefrontal cortex upon the noxious thermally-evoked responses in thalamic centralis lateralis nucleus.

This paper shows a medial prefrontal cortex (CxAP9) facilitating influence upon the unit activity of the centralis lateralis (Cl) nucleus of the thalamus, in rats anesthetized with urethane. Cortical influences were studied using both cortical cooling and cortical spreading depression (CSD) procedures. Both spontaneous and noxious thermally evoked activities were considered. When CSD was propagated and affected the CxAP9, as well as during the cooling of this area, both spontaneous activity and the responses evoked in Cl cells by noxious stimulation were blocked. This effect was interpreted as a cortical disfacilitation upon Cl cells. During the cortical silent period we tested the excitability of a few Cl cells, provoking their activation by passing electrical current across the same Cl recording electrode. No changes were observed in their excitable response threshold during CSD or cortical cooling. Our results are in agreement with the proposition of a tonic cortical facilitatory action upon the spontaneous and noxious-evoked responses recorded in the Cl cells.

Mesencephalic projections to the thalamic centralis lateralis and medial prefrontal cortex: a WGA-HRP study.

In order to provide anatomical information for a possible pathway involved in pain mechanisms, rats were injected with horseradish peroxidase wheat germ agglutinin (WGA-HRP) in the centralis lateralis nucleus of the thalamus (Cl) or in the medial prefrontal cortex (PFCx) from which originated retrogradely labelled cells in the dorsal raphe nucleus (DR), locus ceruleus (LC) and surrounding structures. The locations of the Cl and the PFCx injections were previously determined by the presence of evoked single neuronal responses to noxious stimulations. The present study gives evidence for ascending pathways which originated in DR and LC and project to the Cl and PFCx. LC and DR projections suggest a possible route to an ascending modulation pain system.

Dorsal raphe neuronal responses to thalamic centralis lateralis and medial prefrontal cortex electrical stimulation.

The present study gives evidence for ascending pathways from the dorsal raphe nucleus (DR) to the centralis lateralis nucleus (CL) and to the medial prefrontal cortex (PFCx). Single-unit recordings were done at dorsal raphe level and electrical stimulation was applied at CL and PFCx regions causing antidromic and orthodromic activity in DR cells. The speed conduction difference of the antidromic DR responses to CL and PFCx stimulation was significantly different, but the latencies of the same responses showed no differences. Therefore, we conclude that the DR pathways to CL and to PFCx structures reach their target cells at similar times.

Dorsal raphe and nociceptive stimulations evoke convergent responses on the thalamic centralis lateralis and medial prefrontal cortex neurons.

There is evidence for the existence of a descending pain suppression system, but also there are data supporting the hypothesis for the modulation of pain at higher central nervous system levels. In the present study we give evidence for a possible ascending pain modulation pathway which involves the dorsal raphe (DR), the centralis lateralis nucleus (CL) of the thalamus and the medial prefrontal cortex (PFCx). Urethane-anesthetized rats were used. Simultaneous single unit recordings were done in the CL and PFCx regions under noxious and DR stimulations. Cells responding to both types of stimuli exhibit duration responses directly related to the duration of the stimuli. Thus, from our results we conclude a DR influence upon CL and PFCx structures that are involved in the coding of nociceptive information. A possible route for an ascending pain modulation path is proposed.

Regional brain IR-Met-, IR-Leu-enkephalin concentrations during progress and full electrical amygdaloid kindling.

Using amygdaloid kindling in chronic rats, we were able to observe behavioral, electrographic and IR-Met- and IR-Leu-enkephalin changes throughout the progress of different stages of convulsive activity. Rats presenting the initial stages of kindling, rats presenting the first generalized motor seizure, and rats with at least 10 generalized seizures were sacrificed 24 h after the last stimulus; also rats with at least 10 generalized seizures but sacrificed 21 days after the last seizure were compared with control and sham-operated groups of rats. The IR-Met and IR-Leu enkephalin concentrations in each group were measured in the striatum, amygdala, hypothalamus, medulla oblongata (including pons), hippocampus, mid-brain, spinal cord and cerebral cortex. A progressive increase in IR-Leu-enkephalin in amygdala and hippocampus was observed over the course of kindling. These increases remained until 21 days after rats were fully kindled (at least 10 generalized seizures). We observed increased and decreased concentration of each peptide in different regions. We discussed the regional and the differential effects of each peptide. The increased concentrations in limbic structures were associated with the amygdaloid increased excitability through the kindling process. We suggest that the decreases in concentrations are related with structures involved in the output behavior manifestations produced by kindling stimulation.

Double cortical control acting upon activities of intralaminar thalamic cells.

We report the effects exerted by the cortex upon the intralaminar thalamic nucleic, as revealed by reversible blockade of the cortex with spreading depression in awake rats. Extracellular recordings of spontaneous activity were made simultaneously at thalamic and cortical sites. The effect of peripheral receptive field stimulation was to decrease activity of intralaminar thalamic cells. Cortical recordings revealed the cortical regions affected by spreading depression. Two type of cells were identified depending on the changes in their sensorial responses during the cortical spreading depression propagation. The first exhibited a tonic facilitating cortical control when the cortical spreading depression was located at A 8.0 to A 10.0. The second type exhibited a disappearance of the sensorial responses when cortical spreading depression was located at A 4.0 to A 8.0 and also displayed the tonic facilitating control. This indicates that two different identified cortical regions influenced the thalamic activity.