Biomedical subjects

Kumar N Alagramam

Publications and source records attributed to Kumar N Alagramam.

12 recordsLinked to original sources

PubMed2007

Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.

HYPOTHESIS: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. BACKGROUND: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. METHODS: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. RESULTS: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. CONCLUSION: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Animals↗

PubMed2006

Identification and characterization of mouse cochlear stem cells.

Genetic, noise- and drug-induced loss of hair cells in the mouse and human cochlea leads to permanent hearing loss due to lack of regeneration of hair cells, which may be due to reduced numbers or loss of the regenerative ability of stem cells in the adult cochlea. We hypothesized that the mouse neonate cochlea harbors stem cells capable of differentiating into hair cells. Cells from the primary neonate cochlear culture began to proliferate and formed floating spheres after 14 days in vitro (DIV). By comparison, spheres from the primary culture of the cortex were observed after 7 DIV. Cochlear sphere cells could be passaged and the new spheres were observed after 7 DIV. Cochlear sphere cells were capable of differentiating into astrocytes and oligodendrocytes, but not neurons under the conditions tested. Cochlear sphere cells expressed Sox2 and Myo7a, but failed to show markers that are expressed exclusively in mature cochlear tissue, while cells from cortex spheres expressed Sox2 and Otx2, but not Myo7a. Our results show that cochleae from neonatal mice harbor cells capable of forming spheres and cells from these spheres appear to be better endowed to become hair cells.

Age Factors↗

PubMed2006

N-Acetyl L-cysteine does not protect against premature age-related hearing loss in C57BL/6J mice: a pilot study.

A compound capable of preventing age-related hearing loss would be very useful in an aging population. N-acetyl-L-cysteine (L-NAC) has been shown to be protective against noise exposure, a condition that leads to increased oxidative stress. Not withstanding environmental factors, there is evidence that age-related hearing loss (AHL) in the mouse is linked to more than one genetic loci and, by extension, in humans. Our hypothesis is that AHL defect results in increased sensitivity to oxidative stress and L-NAC would be able to protect the hearing of a mouse model of pre-mature AHL, the C57BL/6J (B6) mouse strain. L-NAC was added to the regular water bottle of B6 mice (experimental group) and available ad lib. The other group received normal tap water. Hearing was tested monthly by the ability to generate the auditory brainstem response (ABR). After the final ABR test, mice were sacrificed by an overdose of Avertin, ears were harvested and hair cell loss was quantified. There was no difference in ABR thresholds or in histopathology between the control group and the group receiving L-NAC in their drinking water. In contrast to the protective effects of L-NAC against noise-induced hearing loss, the lack of protective effect in this study may be due to (i) the dosage level; (ii) the duration of treatment; (iii) the biochemical mechanisms underlying age-induced hearing loss; or (iv) how the mouse metabolizes L-NAC.

Acetylcysteine↗

PubMed2006

Inner ear proteomics of mouse models for deafness, a discovery strategy.

Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.

Animals↗

PubMed2006

Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.

Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.

Animals↗

PubMed2006

Progression of inner ear pathology in Ames waltzer mice and the role of protocadherin 15 in hair cell development.

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 (av-J) and Pcdh15 (av-2J)) and two presumptive functional null alleles (Pcdh15 (av-3J) and Pcdh15 (av-Tg)) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0-P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.

Alleles↗

PubMed2005

Characterization of vestibular dysfunction in the mouse model for Usher syndrome 1F.

The deaf-circling Ames waltzer (av) mouse harbors a mutation in the protocadherin 15 (Pcdh15) gene and is a model for inner ear defects associated with Usher syndrome type 1F. Earlier studies showed altered cochlear hair cell morphology in young av mice. In contrast, no structural abnormality consistent with significant vestibular dysfunction in young av mice was observed. Light and scanning electron microscopic studies showed that vestibular hair cells from presumptive null alleles Pcdh15(av-Tg) and Pcdh15(av-3J) are morphologically similar to vestibular sensory cells from control littermates, suggesting that the observed phenotype in these alleles might be a result of a central, rather than peripheral, defect. In the present study, a combination of physiologic and anatomic methods was used to more thoroughly investigate the source of vestibular dysfunction in Ames waltzer mice. Analysis of vestibular evoked potentials and angular vestibulo-ocular reflexes revealed a lack of physiologic response to linear and angular acceleratory stimuli in Pcdh15 mutant mice. Optokinetic reflex function was diminished but still present in the mutant animals, suggesting that the defect is primarily peripheral in nature. These findings indicate that the mutation in Pcdh15 results in either a functional abnormality in the vestibular receptor organs or that the defects are limited to the vestibular nerve. AM1-43 dye uptake has been shown to correlate with normal transduction function in hair cells. Dye uptake was found to be dramatically reduced in Pcdh15 mutants compared to control littermates, suggesting that the mutation affects hair cell function, although structural abnormalities consistent with significant vestibular dysfunction are not apparent by light and scanning electron microscopy in the vestibular neuroepithelia of young animals.

Animals↗

PubMed2005

A quantitative survey of gravity receptor function in mutant mouse strains.

The purpose of this research was to identify vestibular deficits in mice using linear vestibular evoked potentials (VsEPs). VsEP thresholds, peak latencies, and peak amplitudes from 24 strains with known genetic mutations and 6 inbred background strains were analyzed and descriptive statistics generated for each strain. Response parameters from mutant homozygotes were compared with heterozygote and/or background controls and all strain averages were contrasted to normative ranges. Homozygotes of the following recessive mutations had absent VsEPs at the ages tested: Espn(je), Atp2b2dfw-2J, Spnb4qv-lnd2J, Spnb4qv-3J, Myo7ash1, Tmie(sr), Myo6sv, jc, Pcdh15av-J, Pcdh15av-2J, Pcdh15av-3J, Cdh23v-2J, Sans(js), hr, Kcne1pkr and Pou3f4del. These results suggest profound gravity receptor deficits for these homozygotes, which is consistent with the structural deficits that have been documented for many of these strains. Homozygotes of Catna2cdf, Grid2ho4J, Wnt1sw, qk, and Mbpshi strains and heterozygotes of Grid2lc had measurable VsEPs but one or more response parameters differed from the respective control group (heterozygote or background strain) or were outside normal ranges. For example, qk and Mbpshi homozygotes showed significantly prolonged latencies consistent with the abnormal myelin that has been described for these strains. Prolonged latencies may suggest deficits in neural conduction; elevated thresholds suggest reduced sensitivity, and reduced amplitudes may be suggestive for reduced neural synchrony. One mutation, Otx1jv, had all VsEP response parameters within normal limits--an expected finding because the abnormality in Otxljv is presumably restricted to the lateral semicircular canal. Interestingly, some heterozygote groups also showed abnormalities in one or more VsEP response parameters, suggesting that vestibular dysfunction, although less severe, may be present in some heterozygous animals.

Animals↗

PubMed2005

The basic science of Meniere's disease and endolymphatic hydrops.

PURPOSE OF REVIEW: Meniere's disease is characterized by the triad of fluctuating hearing loss, episodic vertigo, and tinnitus and by endolymphatic hydrops found on post-mortem examination. The cause of Meniere's disease remains unclear. Numerous factors play a role in the development of hydrops and in the pathogenesis of related cochleovestibular dysfunction. This review highlights recent advances in the understanding of the pathophysiology of symptom development in Meniere's disease by detailing the role of genetics, autoimmunity, endolymphatic fluid homeostasis, excitotoxicity, oxidative stress, and cellular apoptosis. Emphasis is placed on reviewing the newly described animal models that exhibit endolymphatic hydrops. RECENT FINDINGS: Recent evidence suggests that hearing loss might be explained in part by apoptosis of spiral ganglion neurons and that hydrops could represent an epiphenomenon rather than an initiating factor. In addition, the accepted guinea pig model described by Kimura has certain limitations. An animal model that would supplement and in some cases replace the surgically induced model is currently being sought. SUMMARY: These recent advances have expanded our understanding and will allow for the development of targeted therapeutic interventions aimed at preventing the progression oochleovestibular deterioration.

Animals↗

PubMed2004

Interactions in the network of Usher syndrome type 1 proteins.

Defects in myosin VIIa, harmonin (a PDZ domain protein), cadherin 23, protocadherin 15 and sans (a putative scaffolding protein), underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. We have previously demonstrated that harmonin interacts with cadherin 23 and myosin VIIa. Here we address the extent of interactions between the five known USH1 proteins. We establish the previously suggested sans-harmonin interaction and find that sans also binds to myosin VIIa. We show that sans can form homomeric structures and that harmonin b can interact with all harmonin isoforms. We reveal that harmonin also binds to protocadherin 15. Molecular characterization of these interactions indicates that through its binding to four of the five USH1 proteins, the first PDZ domain (PDZ1) of harmonin plays a central role in this network. We localize sans in the apical region of cochlear and vestibular hair cell bodies underneath the cuticular plate. In contrast to the other four known USH1 proteins, no sans labeling was detected within the stereocilia. We propose that via its binding to myosin VIIa and/or harmonin, sans controls the hair bundle cohesion and proper development by regulating the traffic of USH1 proteins en route to the stereocilia.

Animals↗

PubMed2003

A new spontaneous mutation in the mouse Ames waltzer gene, Pcdh15.

A recessive deafness mutation in the mouse arose spontaneously and was identified in a colony segregating a null allele of the gastrin-releasing peptide receptor (Grpr) locus. Auditory-evoked brain stem response measurements revealed deafness in 7-week-old affected mice. By linkage analyses, the mutant phenotype was mapped near marker D10Mit186 and the protocadherin gene Pcdh15. As shown by complementation testing, the new mutation is allelic with Ames waltzer (Pcdh15(av)). Sequencing mutant-derived brain Pcdh15 cDNAs identified the insertion of a cytosine residue at nucleotide position c2099 (2099insC), which results in a frame-shift and premature stop codon. Abnormal stereocilia on inner and outer hair cells of the organ of Corti were identified by scanning electron microscopy as early as postnatal day 0 and cross-sectional histology revealed severe neuroepithelial degeneration in cochleas of 30-50-day-old mutants. The new allele of Ames waltzer, designated Pcdh15(av-Jfb), may aid in studying the histopathology associated with Usher syndrome type 1F, which is caused by a functional null allele of PCDH15.

Animals↗

PubMed2003

Assessment of retinal structure and function in Ames waltzer mice.

PURPOSE: In humans, mutations in protocadherin 15 are known to result in Usher Syndrome type 1F (USH1F). Patients with USH1F are born with profound hearing loss and have visual problems that develop in late childhood. Based on the phenotypic hearing loss and an associated mutation in protocadherin 15 (Pcdh15), the Ames waltzer mice have been presented as potential models for USH1F. To determine whether the Ames waltzer is a model for retinopathy in USH1F, retinal structure and function were assessed in all four available alleles of the mouse. METHODS: Activity of both the rod and cone pathways was evaluated by measuring electroretinograms (ERGs) in response to strobe flashes under dark- and light-adapted conditions, respectively. Retinas were processed with standard histochemical procedures, and retinal morphology was examined. The neural retina was dissected from normal pigmented mice at postnatal day (P)0, P5, P7, P20, P40, and P70, and the presence of Pcdh15 was determined by RT-PCR. RESULTS: The amplitude and implicit time of both the rod- and cone-mediated ERG a- and b-waves were comparable between Ames waltzer mutants and heterozygous littermates as old as 13 months. No evidence of retinal degeneration or disorganization was detected in mutant mice. Measures of retinal layer thicknesses were similar in mutant and wild-type control animals. Retinal expression of Pcdh15 was observed at all ages examined between P0 and P70. CONCLUSIONS: Although Pcdh15 is present in neural retina, its role remains unclear. Mutations in the Pcdh15 did not result in retinal abnormalities in the four alleles of Ames waltzer tested in this study. The explanation for the absence of retinal phenotype in the Ames mouse should be helpful in understanding USH1F and developing treatments for this disorder.

Animals↗