John C.M.J de Groot

Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat

To date, most research performed in the area of spinal cord injury focuses on treatments designed to either prevent spreading lesion (secondary injury) or to enhance outgrowth of long descending and ascending fiber tracts around or through the lesion. In the last decade, however, several authors have shown that it is possible to enhance locomotor function after spinal cord injury in both animals and patients using specific training paradigms. As a first step towards combining such training paradigms with pharmacotherapy, we evaluated recovery of function in adult rats sustaining a spinal cord contusion injury (MASCIS device, 12.5 mm at T8), either housed in an enriched environment or in standard cages (n = 15 in both groups). The animals in the enriched environment were stimulated to increase their locomotor activity by placing water and food on opposite sides of the cage. As extra stimuli, a running wheel and several other objects were added to the cage. We show that exposure to the enriched environment improves gross and fine locomotor recovery as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, the BBB subscale, the Gridwalk, and the Thoracolumbar height test. However, no group differences were found on our electrophysiological parameters nor on the amount of spared white matter. These data justify further studies on enriched housing and more controlled exercise training, with their use as potential additive to pharmacological intervention.

Cisplatin-induced ototoxicity: morphological evidence of spontaneous outer hair cell recovery in albino guinea pigs?

Cisplatin is frequently used in the treatment of various forms of malignancies. Its therapeutic efficacy, however, is limited by the occurrence of sensorineural hearing loss. Little is known about the course of hearing loss over longer time intervals after cessation of cisplatin administration. Infrequently, recovery of hearing has been described in animals and humans. Stengs et al. (1997) treated guinea pigs with cisplatin at a daily dose of 1.5 mg/kg for 8 consecutive days and subsequently studied cochlear function after survival times varying from 1 day to 16 weeks. Spontaneous improvement of the hair cell-related potentials (cochlear microphonics and summating potentials) was observed starting 2 weeks after cessation of treatment. In the present study we examined light microscopically the cochleas used in the study of Stengs et al. (1997). One day after cessation of cisplatin administration outer hair cell (OHC) loss in the basal cochlear turn averaged 66%. In the 1-week survival group, OHC counts were similar to those of the 1-day survival group. In the 4-week survival group, however, a relatively small loss of OHCs was found in the basal cochlear turn; OHC loss averaged only 15%. A similar loss was found after 8 weeks. In the 16-week survival group, OHC loss in the basal turn increased to 48%, but this was not statistically significant. Our histological observations are in line with the electrophysiological data from the same animals. Our findings suggest that OHCs recover from cisplatin-induced damage 1-4 weeks after treatment. However, the results do not allow a conclusion as to whether the observed recovery is due to the formation of new OHCs or to (self-)repair of damaged OHCs.

Dose-dependent effect of 8-day cisplatin administration upon the morphology of the albino guinea pig cochlea

Numerous studies investigating cisplatin ototoxicity in animals have been performed, but it is difficult to derive a clear dose-effect relation from these studies. The degree of cisplatin-induced ototoxicity depends on a multitude of factors. Many parameters, such as dose, mode of administration, dosage schedule and concomitant administration of protective additives, vary among the published studies. Therefore, we performed a basic dose-effect study on cisplatin ototoxicity in the guinea pig. Albino guinea pigs were treated with cisplatin at daily doses of either 0.7, 1.0, 1.25, 1.5 or 2.0 mg/kg for 8 consecutive days. Electrocochleography was performed on day 10 after which the cochleas were removed and processed for histological examination. The electrophysiological results showed a marked transition from almost no ototoxic effect to a large effect between a daily dose of 1.25 and 1.5 mg/kg (Stengs et al., 1998). Outer hair cell (OHC) counts corresponded well with the electrophysiological results. At daily doses of 0.7, 1.0 and 1.25 mg/kg no statistically significant OHC loss was observed, whereas OHC loss averaged 60% and 65% in the basal turns at daily doses of 1. 5 and 2.0 mg/kg, respectively. Morphological changes in the stria vascularis were present only in cochleas from animals treated with cisplatin doses of 1.0, 1.25 and 1.5 mg/kg/day. Cochleas from animals treated with a daily cisplatin dose of 2.0 mg/kg for 8 consecutive days showed an endolymphatic hydrops. The present study shows that cisplatin, administered at a daily dose of 1.5 mg/kg for 8 consecutive days, provides a degree of OHC loss that is well suited to study the effects of putative protective agents and possible hair cell recovery.

Protection and Spontaneous Recovery from Cisplatin‐Induced Hearing Loss

ABSTRACT: Cisplatin [cis‐diamminechloroplatinum(II)] has proved itself as a potent antineoplastic agent. However, nephrotoxicity, neurotoxicity, gastrointestinal toxicity, myelosuppression, and ototoxicity interfere with its therapeutical efficacy. Forced diuresis reduces nephrotoxicity, effectively leaving neurotoxicity and ototoxicity as the major side effects of concern, and gastrointestinal toxicity and myelosuppression as the secondary side effects.

Partial recovery of cisplatin-induced hearing loss in the albino guinea pig in relation to cisplatin dose

The objective of the present study was to further characterize cochlear recovery after cisplatin damage. We equipped albino guinea pigs with permanent round window electrodes. Cisplatin was injected i.p. on a daily basis at either 1.5 or 2.0 mg/kg/day. Treatment was stopped when the criterion of > or =40 dB loss in the compound action potential iso-response level at 8 kHz had occurred. Either shortly (1-3 days) or long (4 weeks or more) after this stop, the endocochlear potential (EP) was measured and all animals were sacrificed for histology. At a cisplatin dose of 2.0 mg/kg/day, the time needed to reach the criterion hearing loss varied from 5 to 11 days. With 1.5 mg/kg/day this period lasted longer, the cumulative dose being the first-order predictor. The cochlear potentials gradually recovered in the first 2 weeks after treatment. At the lower frequencies, recovery was often complete. At the higher frequencies complete recovery was never seen. EP was depressed when measured just after treatment but had normal values long after. Basal outer hair cell (OHC) loss was found for both the short and the long post-treatment period. Thus, loss and recovery of cochlear potentials can for a large part be explained by loss and recovery of the EP. Recovery is limited by permanent OHC loss.

Histological effects of co-administration of an ACTH(4–9) analogue, ORG 2766, on cisplatin ototoxicity in the albino guinea pig

Cisplatin is one of the most potent antineoplastic drugs presently known, but its therapeutic efficacy is seriously limited by several side effects such as ototoxicity. Several compounds that are known for their nephroprotective effects also seem to reduce the incidence and severity of cisplatin-induced ototoxicity. Hamers et al. (1994) and De Groot et al. (1997) investigated the possibly protective effect of concomitant administration of the ACTH((4-9)) analogue ORG 2766 upon cisplatin ototoxicity in guinea pigs. Animals were treated with cisplatin at a daily dose of 2.0 mg/kg for 8 consecutive days and ORG 2766 at a daily dose of 75 mcg/kg for 9 days. Concomitant administration of cisplatin plus ORG 2766 resulted in a bimodal distribution of the electrophysiological data (compound action potential and cochlear microphonics amplitudes) and the histological data (outer hair cell (OHC) counts). It was surmised that this dichotomy might occur at a certain cisplatin dose. We investigated whether this protective effect of ORG 2766 could be enhanced by reducing the daily dose of cisplatin while maintaining the same dose of ORG 2766. Thirty-six animals were treated with daily i.p. injections of cisplatin at a dose of 1.0 mg/kg (n=18) or 1.5 mg/kg (n=18) for 8 consecutive days. When comparing the mean OHC counts of the different experimental groups, treatment with cisplatin at a daily dose of 1.5 mg/kg for 8 consecutive days resulted in a considerable loss of OHCs, which was significantly reduced after co-administration of ORG 2766. Co-treatment with ORG 2766 did not result in a change in the volume of the scala media. The present results are in agreement with the electrophysiological results published earlier (Stengs et al., 1998b).

Neurosensory development and cell fate determination in the human cochlea.

BackgroundHearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development.ResultsAt 10xa0weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12xa0weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium prior to hair cell differentiation, and directly targeted the hair cells as they developed. Ubiquitous Peripherin expression by spiral ganglion neurons gradually diminished and became restricted to the type II spiral ganglion neurons by 18xa0weeks. At 20xa0weeks, when the onset of human hearing is thought to take place, the expression profiles in hair cells and spiral ganglion neurons matched the expression patterns of the adult mammalian cochleae.ConclusionsOur study provides new insights into the fetal development of the human cochlea, contributing to our understanding of deafness and to the development of new therapeutic strategies to restore hearing.

Class III β-tubulin, a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.

Perilymphatic application of cisplatin over several days in albino guinea pigs: dose-dependency of electrophysiological and morphological effects.

Cisplatin, at 0, 3, 30 or 300 microg/ml in saline, was applied to the scala tympani of the cochlea of guinea pigs via osmotic mini-pumps, operating at a pump rate of 0.5 microl/h. Electrocochleographic recordings were made from an implanted round window electrode. When an electrocochleographic criterion of ototoxicity was reached (40 dB loss in compound action potential (CAP) threshold at 8 kHz), or after 1 week if this criterion was not reached, the animals were sacrificed for light microscopy. A subgroup of animals had endocochlear potentials (EPs) measured prior to sacrifice. Hearing remained stable in the 0 microg/ml control group, but a sudden drop of auditory sensitivity across the whole frequency range was observed in all other groups. It took 1-5 days before the drop occurred, dependent on cisplatin concentration. CAP and cochlear microphonics were lost simultaneously. The EP was severely depressed in the affected animals, suggesting that cisplatin effects on the EP are primary. However, histology revealed an accompanying loss of outer hair cells, primarily in the basal turn. It is concluded that if cisplatin is given until ototoxicity becomes apparent electrophysiologically, then the cochlear pathology from intrascalar cisplatin administration resembles that from daily parenteral administration at 1.5-2.0 mg/kg. The cochlear pathology from the parenteral treatment was greater than that observed with 30 microg/ml pumps, and less than that from 300 microg/ml pumps.

Experimental Autoimmune Inner Ear Disease: An Electrocochleographic and Histophysiologic Study

Systemic immunization with swine inner ear antigens in complete Freunds adjuvant induces functional disturbances in the cochlea. Morphometric data indicate that an endolymphatic hydrops develops within 2 weeks. It diminishes 6 weeks after immunization. A progressive decrease in the compound action potential amplitude is observed from 2 to 6 weeks after immunization. Enhancement of the amplitude of the summating potential is present without a clear overall correlation to the presence of endolymphatic hydrops. The amplitude of the cochlear microphonics shows no significant changes after immunization. Western blot analysis of the sera performed 2 and 6 weeks after immunization shows enhanced reactivity at 68,50,45, and 27 kd molecular weights, as compared to controls. The same spectrum of cross-reacting antibodies is believed to be instrumental in immune-mediated sensorineural hearing loss in patients. Apparently, cross-reacting antibodies and released mediators disturb cochlear homeostasis, resulting in the observed changes in the electrophysiological responses. However, these changes are not clearly related to structural changes at the light and electron microscopic levels.