Morphological differentiation of bipolar cells in the ferret retina.

Bipolar cells are not only important for visual processing but input from these cells may underlie the reorganization of ganglion cell dendrites in the inner plexiform layer (IPL) during development. Because little is known about the development of bipolar cells, here we have used immunocytochemical markers and dye labeling to identify and follow their differentiation in the neonatal ferret retina. Putative cone bipolar cells were immunoreacted for calbindin and recoverin, and rod bipolar cells were immunostained for protein kinase C (PKC). Our results show that calbindin-immunoreactive cone bipolar cells appear at postnatal day 15 (P15), at which time their axonal terminals are already localized to the inner half of the IPL. By contrast, recoverin-immunoreactive cells with terminals in the IPL are present at birth, but many of these cells may be immature photoreceptors. By the second postnatal week, recoverin-positive cells resembling cone bipolar cells were clearly present, and with increasing age, two distinct strata of immunolabeled processes occupied the IPL. PKC-containing rod bipolar cells emerged by the fourth postnatal week and at this age have stratified arbors in the inner IPL. The early bias of bipolar axonal arbors in terminating in the inner or outer half of the IPL is confirmed by dye labeling of cells with somata in the inner nuclear layer. At P10, several days before ribbon synapses have been previously observed in the ferret IPL, the axon terminals of all dye-labeled bipolar cells were clearly stratified. The results suggest that bipolar cells could provide spatially localized interactions that are suitable for guiding dendritic lamination in the inner retina.

Immunocytochemical localization of GABA, GABAA receptors, and synapse-associated proteins in the developing and adult ferret retina.

Gamma-aminobutyric acid (GABA) modulates the pattern of correlated spontaneous bursting activity between amacrine cells and ganglion cells of the ferret retina during the first postnatal month. Here, we demonstrate the presence of an anatomical network which may underlie these interactions throughout the period when correlated bursting activity is observed, by immunolabelling the neonatal ferret retina for GABA, GABAA receptors, and synapse-associated proteins. GABA immunoreactivity was detected in cell somata in the ganglion cell layer (GCL), in amacrine cells, and in the inner plexiform layer (IPL) by embryonic day 38. This pattern remained largely unchanged throughout neonatal development and in the adult. By contrast to other mammals, the outer plexiform layer (OPL) was only very weakly labelled for GABA, at all ages studied. Strong, punctate, immunolabelling for the beta 2/3 subunit of the GABAA receptor was apparent in the IPL by birth, and appeared in the OPL by the second postnatal week. The possibility that synaptic interactions in the IPL occur during bursting activity was examined by immunolabelling for synapse-associated proteins. Strong immunoreactivity for synaptic vesicle proteins, Synapsin I and II, and synaptic vesicle-2 (SV2), a synaptic vesicle transporter protein, was observed in the IPL by birth. Immunoreactivity for SNAP-25, a protein associated with vesicle fusion, was also intense at the level of the IPL and in the nerve fiber layer of the retina at birth. Taken together, these patterns of immunoreactivity suggest the presence of a GABAergic network in the IPL of the ferret retina by birth, coinciding with the appearance of correlated bursting activity in the inner retina.

Changing patterns of spontaneous bursting activity of on and off retinal ganglion cells during development.

In adult ferrets, retinal ganglion cells (RGCs) responsive to increased (On) or decreased (Off) illumination convey information to different cellular layers of the dorsal lateral geniculate nucleus (dLGN). These dLGN sublaminae emerge during development when RGCs are found to undergo correlated spontaneous bursting activity. Using Ca2+ imaging and intracellular dye-filling techniques, we demonstrate here that in ferret neonates, morphologically identified On and Off beta RGCs have similar burst frequencies prior to the segregation of their inputs in the dLGN, but during the segregation period, they develop distinct burst frequencies. Although the bursts of On cells and Off cells occur synchronously, On cells burst only 25%-35% of the time that Off cells do. This change in the temporal bursting patterns of On and Off RGCs may underlie the segregation of their inputs on dLGN neurons.

Use of sciatic neurogenic motor evoked potentials versus spinal potentials to predict early-onset neurologic deficits when intervention is still possible during overdistraction.

Spinal evoked potentials, sciatic neurogenic motor evoked potentials, and somatosensory evoked potentials were recorded before and after overdistraction of the spinal cord, and compared with the clinical status of 14 pigs. The sciatic neurogenic motor evoked potential consisted of two components: fast and slow. The fast component was more sensitive and associated to a greater degree with motor function in wake-up tests than the slow component somatosensory evoked potential and spinal evoked potential. Furthermore, the loss of only the fast component in the initial status allowed the possibility of improvement of motor activity in the final wake-up test. The peripheral neurogenic motor evoked potentials recording yielded more information about spinal cord function: motor and sensory. The current study suggests that a peripheral response is a better index to the onset of overdistraction and to the efficiency of intervention, when the neurologic deficit after overdistraction of the spine is reversible.

Relationship between duration of spinal cord ischemia and postoperative neurologic deficits in animals.

Stagnara wake-up tests, blood flow measures, somatosensory evoked potentials (SEPs), and neurogenic-motor evoked potentials (NMEPs) were elicited from 20 hogs before and after spinal cord overdistraction at L3-L4. Overdistraction was maintained from 5 to 30 minutes after loss of NMEPs. Results suggest that the longer the duration of overdistraction the greater the likelihood of paraplegia. Blood flow measures indicated that reduced perfusion was greatest at the distraction site but extended proximally and distally. Finally, NMEPs were more sensitive to onset of overdistraction and a more valid indicator of paraplegia than SEPs. NMEPs should provide the surgeon with more time for initiation of intervention techniques than SEPs. Because NMEPs and SEPs provide information regarding different spinal cord tracts, the authors continue to use both methods for monitoring the functional integrity of the human spinal cord during corrective spine surgery.

Blood flow direction in the lumbar nerve root.

The effect of clipping on lumbar nerve root blood flow rates in the region of the nerve root canal was studied experimentally in the hog. Blood flow rate was measured using the hydrogen washout technique. When the entrance zone was clipped with a microvascular clip, blood flow rate of the nerve root was decreased by 37% in comparison with the initial control rate; clipping at the exit zone reduced blood flow rate by 69%. Blood flow direction in the lumbar nerve root within the nerve root canal was found to be predominantly proximal. The current data indicate that the more lateral the impingement of the nerve root occurs, the more ischemic changes are induced.

Relationship between duration of spinal cord ischemia and postoperative neurologic deficits in animals.

Twenty hogs were administered the following procedures before, during, and after overdistraction of the spinal column at T5-T6: somatosensory (SEP) and neurogenic-motor evoked potentials (NMEPs), hydrogen clearance procedures, Stagnara wake-up tests, and aortic-injection of silastic plastic. To ensure that overdistraction was possible, a nonosseous, circumferential osteotomy was made at T5-T6 and distraction applied in one-ratchet increments using Harrington instrumentation. Overdistraction was maintained for 3, 5, 6, 10, 15, 20, 25, or 30 minutes. Results indicated that the duration of overdistraction, as represented by lost NMEPs, was always correlated with the animal's clinical status on wake-up test. If overdistraction was maintained more than 6 minutes, 100% of the animals demonstrated positive wake-up results; if maintained between 5 and 6 minutes, 75% demonstrated positive wake-up results; and if maintained less than 5 minutes, only 25% demonstrated positive wake-up results. Time-to-loss of the NMEPs and SEPs, after onset of overdistraction, fell within two groups: slow and fast. In the slow group, it required slightly more than 20 minutes (mean = 20.6) for the potentials to be lost, while in the fast-loss group data were lost in slightly less than 4 minutes (mean = 3.6). Blood flow studies and inspection of the spinal cord revealed that the mechanism of action for the slow group appeared to be ischemia of the spinal cord that extended several centimeters above and below the site of maximum distraction. In the fast-loss group, it appeared that gross structural damage, with some very localized ischemia, were the mechanisms of actions influencing the integrity of the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ionization on the percutaneous absorption of drugs: partitioning of nicotine into organic liquids and hydrated stratum corneum.

To gain further insight into the disposition of ionizable drugs in the stratum corneum, the partitioning of nicotine as a function of pH was studied in excised, hydrated, human stratum corneum (SC) and the organic liquids n-butanol, n-octanol, isopropyl myristate, and Miglyol 812. Partitioning in n-octanol, isopropyl myristate, and Miglyol 812 was consistent with the pH-partition hypothesis, while partitioning in n-butanol agreed with agreed with a model for the partitioning of the free base and an ion pair which dissociates in the organic phase. The results in SC also suggested the partitioning of ion pairs. Binding studies indicated that neither the un-ionized nor the ionized species is bound significantly in the stratum corneum. Trichloroacetate (TCA) anion increased partitioning of ionized nicotine in n-butanol, but had no effect in stratum corneum. Delipidization of the stratum corneum decreased the partition coefficient for the free base, but had no effect on the ionized species. Thermodynamic parameters determined from van't Hoff plots were consistent with the entry of un-ionized nicotine into ordered lipids. These results suggest that the un-ionized form is found within the lipid regions of the stratum corneum, while the ionized form is located in aqueous regions.

Technical factors affecting the reproducibility of intravesical mouse bladder tumor implantation during therapy with Bacillus Calmette-Guérin.

Four methods of intravesical implantation of the transplantable mouse bladder tumor, MBT-2, and their effects on intravesical therapy with Bacillus Calmette-Guérin (BCG) were compared, and modifications which improved implantation are described. Pretreatment of the bladder with N-methyl-N-nitrosourea (MNU) resulted in tumor implantation in approximately two-thirds of the animals; however, all tumors penetrated the bladder wall. Using the MNU implantation procedure, intravesical BCG therapy was shown to reduce MBT-2 outgrowth by 77%. Tumor cell instillation after electrocautery produced an incidence of tumor implantation similar to that of the MNU procedure. The efficacy of BCG for the electrocautery implantation procedure also was similar to the MNU method. With the electrocautery procedure, the electrode and tumor cells were introduced into the bladder via a catheter prepared from PE 10 polyethylene tubing. The procedure required two catheterizations and produced a 24% incidence of extravesical tumors. Use of a Teflon catheter and a single catheterization for tumor cell instillation resulted in a reproducible method for implanting MBT-2 tumors which were all confined within the bladder. The efficacy of BCG therapy was unchanged from that described for the other implantation techniques.

Comparison of the efficacy of intravesical Bacillus Calmette-Guérin with thiotepa, mitomycin C, poly I:C/poly-L-lysine and cis platinum in murine bladder cancer.

The efficacy of intravesical Bacillus Calmette-Guérin for the treatment of the mouse bladder tumor MBT-2 was compared with that of thiotepa, mitomycin C, cis-diamminedichloroplatinum II and poly I:C/poly-L-lysine. MBT-2 cells were instilled into the bladder immediately after electrocauterization. Drug instillations were initiated 24 hours later and continued on a weekly basis for 4 weeks. Both Bacillus Calmette-Guérin and cis-diamminedichloroplatinum II significantly (p less than .0004) inhibited MBT-2 tumor implantation when compared to diluent-treated controls. Neither mitomycin C, thiotepa nor poly I:C/poly-L-lysine significantly inhibited tumor implantation. Mean tumor weights also were significantly (p less than .05) reduced in Bacillus Calmette-Guérin and cis-diamminedichloroplatinum II-treated mice, while tumor mean weights in mice treated with thiotepa, mitomycin C or poly I:C/poly-L-lysine were not significantly different than controls. These results suggest that the efficacy of intravesical Bacillus Calmette-Guérin in comparison with other drugs in the MBT-2 mouse bladder tumor model is similar to observations reported in human clinical trials in which intravesical Bacillus Calmette-Guérin was shown to be more effective than other cytotoxic drugs. These data further support the utility of the MBT-2 model for the study of the mechanisms by which Bacillus Calmette-Guérin inhibits bladder tumor growth.

Reduction of bladder tumor growth in mice treated with intravesical Bacillus Calmette-Guérin and its correlation with Bacillus Calmette-Guérin viability and natural killer cell activity.

The effect of intravesical Bacillus Calmette-Guérin (BCG; Pasteur strain) treatment on the frequency of implantation and growth rate of the murine transitional cell carcinoma of the bladder, MBT-2, was studied. MBT-2 cells were instilled into the bladder immediately after electrocauterization, and BCG instillations (40, 80, and 160 micrograms/instillation) were initiated 24 hr later and continued on a weekly basis for 4 weeks. BCG treatment significantly (p less than 0.0002) reduced the incidence of tumor implantation in a dose-dependent manner and resulted in significantly (p less than 0.0001) smaller tumors when they appeared in BCG-treated mice. The therapeutic effect of BCG correlated with augmentation of natural killer cell (NK) activity and positive purified protein derivative (PPD) footpad reactions. In experiments in which treatment was initiated with rapidly growing BCG organisms (10(7) colony-forming units/mg), tumor implantation was inhibited, there was a dose-dependent increase in NK activity, and mice had positive footpad reactions in PPD. In experiments in which BCG with reduced viability (10(6) colony-forming units/mg) and slower growth rates was used for treatment, no significant inhibition of tumor implantation was observed, NK activity was depressed, and PPD footpad tests were uniformly negative. The results suggest that the therapeutic effects of BCG therapy in this murine model correlate with augmentation of NK activity and positive footpad reactions to PPD and further suggest that the viability and growth rate of BCG organisms are important factors in determining the efficacy of intravesical BCG therapy.

Electric birefringence evaluation of particle size distributions: theory for polydisperse equivalent spheres.

Electric fields induce orientational order in particulate suspensions thereby rendering them birefringent. The decay rate of the birefringence following the field termination is characteristic of sample size, shape, and polydispersity. Theory is developed herein for the evaluation of a two-parameter function for the distribution of sizes of arbitrarily shaped particles in terms of their equivalent spherical diameters. An experimental procedure, involving the measurement of the birefringence decay characteristics in two well-defined experimental conditions, is outlined and used to check the theory for a colloidal suspension of polytetrafluoro-ethylene particles in water.