A partial hearing animal model for chronic electro-acoustic stimulation.

OBJECTIVE: Cochlear implants (CIs) have provided some auditory function to hundreds of thousands of people around the world. Although traditionally carried out only in profoundly deaf patients, the eligibility criteria for implantation have recently been relaxed to include many partially-deaf patients with useful levels of hearing. These patients receive both electrical stimulation from their implant and acoustic stimulation via their residual hearing (electro-acoustic stimulation; EAS) and perform very well. It is unclear how EAS improves speech perception over electrical stimulation alone, and little evidence exists about the nature of the interactions between electric and acoustic stimuli. Furthermore, clinical results suggest that some patients that undergo cochlear implantation lose some, if not all, of their residual hearing, reducing the advantages of EAS over electrical stimulation alone. A reliable animal model with clinically-relevant partial deafness combined with clinical CIs is important to enable these issues to be studied. This paper outlines such a model that has been successfully used in our laboratory. APPROACH: This paper outlines a battery of techniques used in our laboratory to generate, validate and examine an animal model of partial deafness and chronic CI use. MAIN RESULTS: Ototoxic deafening produced bilaterally symmetrical hearing thresholds in neonatal and adult animals. Electrical activation of the auditory system was confirmed, and all animals were chronically stimulated via adapted clinical CIs. Acoustic compound action potentials (CAPs) were obtained from partially-hearing cochleae, using the CI amplifier. Immunohistochemical analysis allows the effects of deafness and electrical stimulation on cell survival to be studied. SIGNIFICANCE: This animal model has applications in EAS research, including investigating the functional interactions between electric and acoustic stimulation, and the development of techniques to maintain residual hearing following cochlear implantation. The ability to record CAPs via the CI has clinical direct relevance for obtaining objective measures of residual hearing.

Cochlear implantation for chronic electrical stimulation in the mouse.

The mouse is becoming an increasingly attractive model for auditory research due to the number of genetic deafness models available. These genetic models offer the researcher an array of congenital causes of hearing impairment, and are therefore of high clinical relevance. To date, the use of mice in cochlear implant research has not been possible due to the lack of an intracochlear electrode array and stimulator small enough for murine use, coupled with the difficulty of the surgery in this species. Here, we present a fully-implantable intracochlear electrode stimulator assembly designed for chronic implantation in the mouse. We describe the surgical approach for implantation, as well as presenting the first functional data obtained from intracochlear electrical stimulation in the mouse.

Chronic electrical stimulation of the auditory nerve at high stimulus rates: a physiological and histopathological study.

A major factor associated with recent improvements in the clinical performance of cochlear implant patients has been the development of speech-processing strategies based on high stimulation rates. While these processing strategies show clear clinical advantage, we know little of their long-term safety implications. The present study was designed to evaluate the physiological and histopathological effects of long-term intracochlear electrical stimulation using these high rates. Thirteen normal-hearing adult cats were bilaterally implanted with scala tympani electrode arrays and unilaterally stimulated for periods of up to 2100 h using either two pairs of bipolar or three monopolar stimulating electrodes. Stimuli consisted of short duration (25-50 microseconds/phase) charge-balanced biphasic current pulses presented at 1000 pulses per second (pps) per channel for monopolar stimulation, and 2000 pps/channel for bipolar stimulation. The electrodes were shorted between current pulses to minimize any residual direct current, and the pulse trains were presented using a 50% duty cycle (500 ms on; 500 ms off) in order to simulate speech. Both acoustic (ABR) and electrical (EABR) auditory brainstem responses were recorded periodically during the chronic stimulation program. All cochleas showed an increase in the click-evoked ABR threshold following implant surgery; however, recovery to near-normal levels occurred in approximately half of the stimulated cochleas 1 month post-operatively. The use of frequency-specific stimuli indicated that the most extensive hearing loss generally occurred in the high-frequency basal region of the cochlea (12 and 24 kHz) adjacent to the stimulating electrode. However, thresholds at lower frequencies (2, 4 and 8 kHz), appeared at near-normal levels despite long-term electrode implantation and electrical stimulation. Our longitudinal EABR results showed a statistically significant increase in threshold in nearly 40% of the chronically stimulated electrodes evaluated; however, the gradient of the EABR input/output (I/O) function (evoked potential response amplitude versus stimulus current) generally remained quite stable throughout the chronic stimulation period. Histopathological examination of the cochleas showed no statistically significant difference in ganglion cell densities between cochleas using monopolar and bipolar electrode configurations (P = 0.67), and no evidence of cochlear damage caused by high-rate electrical stimulation when compared with control cochleas. Indeed, there was no statistically significant relationship between spiral ganglion cell density and electrical stimulation (P = 0.459), or between the extent of loss of inner (IHC, P = 0.86) or outer (OHC, P = 0.30) hair cells and electrical stimulation. Spiral ganglion cell loss was, however, influenced by the degree of inflammation (P = 0.016) and electrode insertion trauma. These histopathological findings were consistent with the physiological data. Finally, electrode impedance, measured at completion of the chronic stimulation program, showed close correlation with the degree of tissue response adjacent to the electrode array. These results indicated that chronic intracochlear electrical stimulation, using carefully controlled charge-balanced biphasic current pulses at stimulus rates of up to 2000 pps/channel, does not appear to adversely affect residual auditory nerve elements or the cochlea in general. This study provides an important basis for the safe application of improved speech-processing strategies based on high-rate electrical stimulation.

Cochlear pathology following chronic electrical stimulation of the auditory nerve. I: Normal hearing kittens.

The present study examines the histopathological effects of long-term intracochlear electrical stimulation in young normal hearing animals. Eight-week old kittens were implanted with scala tympani electrode arrays and stimulated for periods of up to 1500 h using charge balanced biphasic current pulses at charge densities in the range 21-52 microC cm-2 geom. per phase. Both click and electrically evoked auditory brainstem responses were periodically recorded to monitor the status of the hair cell and spiral ganglion cell populations. In addition, the impedance of the stimulating electrodes was measured daily to monitor their electrical characteristics during chronic implantation. Histopathological examination of the cochleas showed no evidence of stimulus induced damage to cochlear structures when compared with implanted, unstimulated control cochleas. Indeed, there was no statistically significant difference in the ganglion cell density adjacent to the stimulating electrodes when compared with a similar population in implanted control cochleas. In addition, hair cell loss, which was restricted to regions adjacent to the electrode array, was not influenced by the degree of electrical stimulation. These histopathological findings were consistent with the evoked potential recordings. Finally, electrode impedance data correlated well with the degree of tissue growth observed within the scala tympani. The present findings indicate that the young mammalian cochlea is no more susceptible to cochlear pathology following chronic implantation and electrical stimulation than is the adult.

A gated differential amplifier for recording physiological responses to electrical stimulation.

Artifact from electrical stimulation imposes a problem for the recording of physiological responses to electrical stimulation. Here we describe a simple, low-cost, gated differential amplifier for the recording of physiological responses to electrical stimulation. The gain of the amplifier is set to 1 during electrical stimulation by setting the gate input to a high logic state to avoid overloading of the amplifier by the artifact. Following electrical stimulation, the gate input is set to a low logic state, resulting in a gain of 1000 for frequencies between 300 Hz and 25 kHz (-3 dB points). The gain at low frequencies (0-0.2 Hz) is held constant at 1 to avoid transients in the output signal arising from changes in gain at these frequencies. The gain of the amplifier following stimulation (gate low) was independent of the magnitude of the artifact and was therefore suitable for the measurement of neural field potentials with low impedance electrodes.

Cochlear pathology following chronic electrical stimulation using non charge balanced stimuli.

During the course of a chronic intracochlear electrical stimulation study using charge balanced biphasic current pulses, one animal inadvertently received a short period of direct current (DC) stimulation at a level of approximately 1 microA. Subsequent, the animal was chronically stimulated using a poorly charge balanced waveform that produced a DC level of approximately 2 microA. Extensive pathological changes were observed within the cochlea. These changes included widespread spiral ganglion cell loss and new bone growth that extended throughout all turns of the cochlea. Significant changes in the morphology of the electrically evoked auditory brainstem response (EABR) were associated with these pathological changes. EABRs recorded prior to the DC stimulation exhibited a normal waveform morphology. However, responses recorded during the course of the DC stimulation were dominated by a short latency response believed to be vestibular in origin. The response thresholds were also significantly higher than levels recorded before the DC stimulation. In contrast, the contralateral cochlea, stimulated using charge balanced stimuli, showed no evidence of adverse pathological changes. Furthermore, EABRs evoked from this cochlea remained stable throughout the chronic stimulation period. Although preliminary, the present results illustrate the adverse nature of poorly charge balanced electrical stimuli. These results have important implications for both the design of neural prostheses and the use of DC stimuli to suppress tinnitus in patients.

Functional studies of cell-mediated immunity in haemophilia and other bleeding disorders.

Defects of immunoregulation which occur in haemophilia, reflected by numerical changes in T lymphocyte subsets, have been further investigated in functional studies. Polyclonal T-cell activation by the mitogen phytohaemagglutinin (PHA) and specific stimulation by cytomegalovirus (CMV) or herpes simplex type 2 (HSV-2) in previously sensitized subjects were studied in peripheral blood lymphocyte and in T4-cell-enriched cultures. Compared with 12 controls, the responses of 11 patients (nine with haemophilia A and two with von Willebrand's disease) to PHA were significantly reduced both in unfractionated and in T4-cell-enriched peripheral blood lymphocyte cultures. Reduced responses to PHA were found in HIV (HTLV III)-seronegative as well as -seropositive patients. There were no significant differences between the response of patients' unfractionated and T4-enriched peripheral blood lymphocytes to CMV/HSV-2 recall antigen and the control subjects, although there was evident variation in the magnitude of patients' unfractionated and T4-enriched lymphocyte responses.

Expression of functional interleukin 2 receptors on chronic lymphocytic leukaemia B lymphocytes is modulated by recombinant interleukin 2.

In 16 of 18 chronic lymphocytic leukaemia (CLL) patients examined, a significant proportion of B cells in the leukaemic clone bound monoclonal antibodies specific for the interleukin 2 (IL-2) receptor site (CD25). B lymphocytes from patients tested showed a direct response to recombinant interleukin (rIL-2) during culture in vitro as shown by: (a) a ligand-mediated upregulation in the level of IL-2 receptor (IL-2R) expression (12 of 12 patients), (b) an increase in cell size (eight of nine patients), (c) an increase in 3H-thymidine uptake (four of six patients). Taken together, this evidence suggests that the majority of leukaemic B cells from all the CLL patients examined expressed functional IL-2 receptors in vitro. Intriguingly, maximal receptor upregulation or increase in cell size was achieved at a lower concentration (50 u/ml) of rIL-2 than was required to achieve maximal 3H-thymidine incorporation.

Analysis and isolation of leukaemic and residual normal B lymphocyte populations from patients with chronic lymphocytic leukaemia.

Two colour fluorescent staining using anti-Leu-1 and anti-B1 monoclonal antibodies has been combined with continuous flow microfluorimetry to analyse and isolate the T and B cell populations in B cell chronic lymphocytic leukaemia (B-CLL) patients. In Rai stage 0 patients two distinct B1 positive populations were detected and sorted; one was Leu-1- and the other Leu-1+. Analysis of the isolated populations showed that the former (Leu-1- B1+) represented the residual normal B cells, whereas the latter represented monoclonal CLL B cells. Short term culture of these cells in vitro showed that the Leu-1+ B1+ population secreted IgM, but not IgG, following PMA stimulation.

Serial studies of T-lymphocyte subpopulations in HIV seropositive haemophiliacs.

Serial determinations of the numbers of peripheral blood T-lymphocyte subpopulations (T-helper/inducer and T-suppressor/cytotoxic cells) were made using monoclonal antibodies in 12 patients with haemophilia who were HIV antibody positive. Eleven patients were clinically severe and one clinically moderate. Follow-up studies for 12-42 months in nine patients showed persisting abnormalities in five patients, abnormalities which developed in three patients who were initially normal, and a return to normal findings in one patient who was initially abnormal. The remaining three patients were followed up for 3-4 months; two showed persisting abnormalities and one a return to normal. Persistent generalized lymphadenopathy was found in one patient who showed raised levels of T-suppressor cells initially and low levels of T-helper cells in the follow-up, but low T-helper cells persisting for up to 12 months were not necessarily associated with disease.

Quantitation of immunoglobulin isotypes in chronic lymphocytic leukaemic cells compared with normal adult and neonatal lymphocytes.

Radioimmunoassays were used to measure the total cellular content of mu, gamma, alpha, delta, kappa and lambda immunoglobulin chains in peripheral blood lymphocytes from normal adults, neonates and 11 patients with chronic lymphocytic leukaemia (CLL). Normal adult lymphocytes contained all classes of immunoglobulin, but predominantly IgG, associated with both types of light chain (kappa:lambda ratio 2:1). In contrast, mu was the major heavy chain in cells from 10 of the CLL patients, and the small amount of IgG found in CLL cells was not produced by the leukaemic clone. Approximately equimolar amounts of one type of light chain were also present, indicating monoclonality. The class distribution of the immunoglobulin in the neonatal cells was intermediate between that of CLL and normal adult cells. CLL B cells had substantially less surface IgM than normal but more cytoplasmic IgM. These data demonstrate the immaturity of neonatal B cells and suggest that CLL cells are also immature--at a stage not normally found in the adult circulation.

Disturbances in T-cell subpopulations in B-cell chronic lymphocytic leukaemia.

The relative proportions of immunoregulatory T-cell subpopulations defined by OKT monoclonal antibodies are disturbed in chronic lymphocytic leukaemia (CLL) patients (even early mild cases) compared with normal subjects in the same age range. The mean ratio OKT4+:OKT8+ (helper:suppressor/cytotoxic) is reversed from the normal 1.6:1 to 1:1.9. The absolute concentration of each OKT population in the circulation is slightly higher than normal, the increase in OKT8+ being the most significant. The E-rosette-forming cells did not always correlate with OKT+ cells and, in four cases, the discrepancy between the size of the OKT3+ population and the sum of OKT4+ and OKT8+ suggested the presence of T cells with an immature (thymic) phenotype in peripheral blood. These abnormalities may account for the depressed immune function of CLL patients.

Mitogen-stimulated immunoglobulin production by chronic lymphocytic leukaemic lymphocytes.

The capacity of B cells from patients with chronic lymphocytic leukaemia (CLL) to produce and secrete immunoglobulin following mitogen stimulation was investigated using sensitive radioimmunoassays for mu, gamma, alpha, kappa, and lambda immunoglobulin chains. Lymphocytes from seven of the 11 patients studied secreted immunoglobulin in response to pokeweed mitogen (PWM). IgM was always the major immunoglobulin and in five of the seven responders it was the only class detected; only one type of light chain was observed in most cases. This was in contrast to normal lymphocytes which secreted all classes of immunoglobulin (IgM was invariably the lowest) containing both types of light chain. Lipopolysaccharide induced immunoglobulin secretion in only one of four CLL cases. This was again IgM with only one type of light chain. The assays are therefore most probably measuring a response by the leukaemic cells. In most CLL cases, immunoglobulin secretion by the residual normal cells, which proliferate in response to mitogen, was not observed. This inability of the normal lymphocytes to differentiate fully into immunoglobulin secreting cells and the block in switching from IgM production to other classes in the leukaemic cells may both be attributable to a defect in the regulatory system of the immune response in CLL patients.

The roles of leukaemic and residual normal cells in the proliferative response of chronic lymphocytic leukaemic lymphocytes to mitogens.

The proliferative response of lymphocytes from patients with chronic lymphocytic leukaemia (CLL) to the polyclonal activators, phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) correlates inversely with the logarithm of the circulating lymphocyte concentration in vivo (P less than 0.001). This suggests that the response is due primarily to residual normal cells in the circulation. In support of this postulate, dilution of normal cells reproduced the effect and also induced a delayed response to PHA, which is found frequently with CLL lymphocytes. A combination of autoradiography and immunofluorescence microscopy identified both B and T cells in the responding population in similar proportions from both normal and CLL donors. These data demonstrate that the 3H-thymidine assay used in most mitogenic studies is not suitable alone for investigating the functional capacity of leukaemic lymphocytes in CLL and other diseases involving a gross perturbation of lymphocyte populations.

Studies of T, B, and "null" blood lymphocytes in normal persons of different age groups.

The numbers of T, B, and "null" blood lymphocytes were determined in 216 normal healthy persons, whose ages ranged from 20 to 94 years. No change was found in the numbers of T or B lymphocytes with increasing age of the subjects, whereas both the total lymphocytes and the number of "null" lymphocytes decreased just significantly with age (p less than 0.05). The response of blood lymphocytes to phytohaemagglutinin in vitro was measured in 25 subjects aged 20-30 years and compared with the response in 23 subjects aged over 60 years. There was no significant difference in the response of the two groups.

Radiosensitivity and recovery of two murine haemopoietic progenitor cell populations following gamma rays and neutrons.

Experiments measuring the sensitivity of mouse bone marrow stem cells (CFU-S) and granulopoietic progenitor cells (CFU-C) to gamma-rays and neutrons are described. Both populations are more sensitive to neutrons than to gamma-rays, and in each case CFU-S are more sensitive than CFU-C. The CFU-C (but not CFU-S) show a biphasic dose-response curve to gamma-rays, and the data suggest that 20-50% of CFU-C in normal mice are more resistant to gamma-rays than the rest, possibly due to hypoxia. This difference between the two cell populations may be related to differences in spatial location in the bone marrow. The recovery of these cells following gamma-rays is consistent with previous data which suggests that CFU-C are not self-maintaining, but require to be fed in from the stem cells.

Changes in T and B blood lymphocytes after splenectomy.

The blood lymphocytes of 37 splenectomised patients were analysed by means of T and B lymphocyte surface markers. Sixteen patients had had a splenectomy for non-haematological and 21 for haematological reasons. The results show that 15 had normal numbers of T and B cells; decreased T cells were found in two patients, raised B cells in seven, raised T and B cells in eight, and raised T cells in five patients. Increased numbers of 'null' cells were observed in some patients, especially in those with raised B cells. Follow-up studies indicate that raised levels of T and B cells can be established by one to three months post-splenectomy and may persist, although in some patients the cells fall to normal levels. The lymphocyte proliferative response to phytohaemagglutinin and Concanavalin A in vitro was normal in eight out of nine patients with raised T cells and was depressed in one patient, possibly due to an intrinsic cell defect.

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents.

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous gamma-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after fusulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells ("committed" stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.