A comparative analysis of cone photoreceptor morphology in bowhead and beluga whales.

The cetacean visual system is a product of selection pressures favoring underwater vision, yet relatively little is known about it across taxa. Previous studies report several mutations in the opsin genetic sequence in cetaceans, suggesting the evolutionary complete or partial loss of retinal cone photoreceptor function in mysticete and odontocete lineages, respectively. Despite this, limited anatomical evidence suggests cone structures are partially maintained but with absent outer and inner segments in the bowhead retina. The functional consequence and anatomical distributions associated with these unique cone morphologies remain unclear. The current study further investigates the morphology and distribution of cone photoreceptors in the bowhead whale and beluga retina and evaluates the potential functional capacity of these cells' alternative to photoreception. Refined histological and advanced microscopic techniques revealed two additional cone morphologies in the bowhead and beluga retina that have not been previously described. Two proteins involved in magnetosensation were present in these cone structures suggesting the possibility for an alternative functional role in responding to changes in geomagnetic fields. These findings highlight a revised understanding of the unique evolution of cone and gross retinal anatomy in cetaceans, and provide prefatory evidence of potential functional reassignment of these cells.

Whale tear glands in the bowhead and the beluga whales: Source and function.

Orbital glands are found in many tetrapod vertebrates, and are usually separate structures, consisting of individual glands lying in the eyelids and both canthi of the orbit. In cetaceans, however, the orbital glandular units are less distinct and have been described by numerous authors as a single, periorbital mass. There are few histochemical and immunhistochemical studies to date of these structures. In this study, we examined the orbital glandular region of both the bowhead whale (Balaena mysticetus: Mysticeti) and the beluga whale (Delphinapterus leucas: Odontoceti) using histological, histochemical, and immunohistochemical techniques. Histologically, in the bowhead, three glandular areas were noted (circumorbital, including Harderian and lacrimal poles), palpebral (midway in the lower eyelid), and rim (near the edge of the eyelid). In the beluga, there was only a large, continuous mass within the eyelid itself. Histochemical investigation suggests neither sexual dimorphism nor age-related differences, but both whales had two cell types freely intermingling with each other in all glandular masses. Large cells (cell type 1) were distended by four histochemically distinct intracellular secretory granules. Smaller cells (cell type 2) were not distended (fewer granules) and had two to three histochemically distinct intracellular secretory granules. The beluga orbital glands had additional lipid granules in cell type 1. Counterintuitively, both lipocalin and transferrin were localized to cell type 2 only. This intermingling of cell types is unusual for vertebrates in whom individual orbital glands usually have one cell type with one to two different secretory granules present. The heterogeneity of the orbital fluid produced by cetacean orbital glands implies a complex function, or series of functions, for these orbital glands and their role in producing the tear fluid.

The role of desmosomes in the ear plug formation in the bowhead whale (Balaena mysticetus).

The external acoustic meatus (EAM) of most baleen whales accumulates cellular debris annually in the lumen as whales age, forming a lamellated ear plug. The bowhead whale ear plug is formed from annually molting lining of the EAM as the entire epithelium releases at the level of the stratum basale during the spring migration. Epithelial regeneration is mostly completed by the fall migration, remaining intact for 6-7 months before being torn off the following spring. Desmosomes are integral to cell-cell adhesion with connecting desmosomal cadherins desmoglein (dsg) and desmocollin (dsc). Paraffin sections of the oral cavity and EAM lining of spring and fall adult bowhead whales, as well as the EAM of spring-caught juvenile, were immunohistochemically examined for the presence of these cadherins. In all fall specimens, both cadherins occurred in all layers except the superficial keratinous layer of the oral cavity. In spring, three different conditions existed: (a) oral cavity of spring-caught adults had reduced cadherins, with superficial fissuring in its keratinized layer and vacuolation in the upper stratum spinosum; (b) EAM of juvenile spring-caught whales displayed fissuring with accompanying reduction of both cadherins in its superficial lining; and (c) EAM lining of spring-caught adults displayed deep fissures, reduced cadherins, and absence of dsc1 in the fissuring zone. These results suggest that shedding of skin layers in mammals, whether normal molting, pathological, or the result of injury and wound repair all revolve around desmosome function. The specific role, structure, and location of these two cadherins need to be further addressed.

Structure of the external auditory meatus of the Bowhead whale (Balaena mysticetus) and its relation to their seasonal migration.

The external auditory meatus (EAM) in many species of mysticete whales is filled with a waxy ear plug. Though this lamellated structure is often used to age a whale, its formation and development remain undescribed. It is thought that growth layer groups (GLGs) are laid down annually, thereby increasing the size of this structure. Since some mysticete whales are migratory and many undergo molting, we hypothesized that the cyclical production of these GLGs may be related to these processes. The epithelia of both EAM and glove finger (a part of the tympanic membrane protruding into the EAM) of one juvenile and multiple adult bowhead whales from both fall (October: non-molting) and spring (May: molting) seasons were dissected and examined anatomically and histologically. These tissue samples were compared with the adult oral epithelia at the same time periods. These epithelia shared a similar basic broad structure, though there were differences in thickness and presence of intraepithelial structures. All epithelia in the October specimens were rich in both glycogen and lipid. The parakeratinized epithelium of the oral cavity in the juvenile and some May specimens shed via the production of several superficial epithelial fissures. Other adult May specimens exhibited deep epithelial fissures, reminiscent of pressure ulcers, which would cause the detachment of the entire epithelium from the dermis. We propose that sloughed epithelial lining is the source of the GLGs in the ear plug. Correlating a potential molting sequence with these observations explained the presence of epidermal glycogen, deep epidermal fissures and dermal glycolipid, and to some extent calls into question the origin and structure of the ear plug itself. Further morphological characterization of ear plugs in bowheads is needed to better understand cell origin and ear plug formation.

Evolutionary aspects of the development of teeth and baleen in the bowhead whale.

In utero, baleen whales initiate the development of several dozens of teeth in upper and lower jaws. These tooth germs reach the bell stage and are sometimes mineralized, but toward the end of prenatal life they are resorbed and no trace remains after birth. Around the time that the germs disappear, the keratinous baleen plates start to form in the upper jaw, and these form the food-collecting mechanism. Baleen whale ancestors had two generations of teeth and never developed baleen, and the prenatal teeth of modern fetuses are usually interpreted as an evolutionary leftover. We investigated the development of teeth and baleen in bowhead whale fetuses using histological and immunohistochemical evidence. We found that upper and lower dentition initially follow similar developmental pathways. As development proceeds, upper and lower tooth germs diverge developmentally. Lower tooth germs differ along the length of the jaw, reminiscent of a heterodont dentition of cetacean ancestors, and lingual processes of the dental lamina represent initiation of tooth bud formation of replacement teeth. Upper tooth germs remain homodont and there is no evidence of a secondary dentition. After these germs disappear, the oral epithelium thickens to form the baleen plates, and the protein FGF-4 displays a signaling pattern reminiscent of baleen plates. In laboratory mammals, FGF-4 is not involved in the formation of hair or palatal rugae, but it is involved in tooth development. This leads us to propose that the signaling cascade that forms teeth in most mammals has been exapted to be involved in baleen plate ontogeny in mysticetes.

ECM production of primary human and bovine chondrocytes in hybrid PEG hydrogels containing type I collagen and hyaluronic acid.

The development of advanced materials that facilitate hyaline cartilage formation and regeneration in aging populations is imperative. Critical to the success of this endeavor is the optimization of ECM production from clinically relevant cells. However, much of the current literature focuses on the investigation of primary bovine chondrocytes from young calves, which differ significantly than osteoarthritic cells from human sources. This study examines the levels of extracellular matrix (ECM) production using various levels of type I collagen and hyaluronic acid in poly(ethylene glycol) dimethacrylate (PEGDM) hydrogels in total knee arthroplasties, compared with the results from bovine chondrocytes. The addition of type 1 collagen in both the presence and absence of low levels of hyaluronic acid increased ECM production and/or retention in scaffolds containing either bovine or human chondrocytes. These findings are supported consistently with colorimetric quantification, whole mount extracellular matrix staining for both cell types, and histological staining for glycoaminoglycans and collagen of human chondrocyte containing samples. While exhibiting similar trends, the relative ECM productions levels for the primary human chondrocytes are significantly less than the bovine chondrocytes which reinforces the need for additional optimization.

The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress.

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.

Characterization of ex vivo-generated bovine and human cartilage by immunohistochemical, biochemical, and magnetic resonance imaging analyses.

Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 microL spot cultures with 5 x 10(6) each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo-formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo-formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.

Advanced osteoarthritis in humans is associated with altered collagen VI expression and upregulation of ER-stress markers Grp78 and bag-1.

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen.

A novel role for Bcl-2 associated-athanogene-1 (Bag-1) in regulation of the endoplasmic reticulum stress response in mammalian chondrocytes.

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.

Ossification of the mouse metatarsal: differentiation and proliferation in the presence/absence of a defined growth plate.

There is significant diversity in growth plate behavior among sites within an individual skeleton and between skeletons of different species. This variation within wild-type animals is an underutilized resource for studying skeletal development. One bone that potentially exhibits the most diverse behavior is the metatarsal. While one end forms a growth plate with an epiphyseal secondary center of ossification as in other long bones, the opposite end undergoes direct ossification in a manner more similar to short bones. Although descriptions of human metatarsal/metacarpal ossification are available, a detailed comparative analysis has yet to be conducted in an animal model amenable to biomolecular analysis. Here we report an analysis of proximal and distal ossification in an age series of mouse metatarsals. Safranin O staining was used for qualitative and quantitative histology, and chondrocyte differentiation and proliferation were analyzed using immunohistochemistry for type X collagen and proliferative cell nuclear antigen expression. We establish that, as in the human, both growth plate formation and direct ossification occur in the mouse metatarsal, with chondrocyte populations showing distinct differentiation patterns at opposite ends of the bone. In addition, growth plate formation is characterized by a peak of proliferation in reserve zone chondrocytes that distinguishes it from both established growth plates and direct ossification. Our analysis demonstrates that the mouse metatarsal is a productive model for investigating natural variation in ossification that can further understanding of vertebrate skeletal development and evolution.

Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis.

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.

Increased chondrocyte apoptosis in growth plates from children with slipped capital femoral epiphysis.

Ultrastructural studies of slipped capital femoral epiphysis (SCFE) growth plates have shown diminished cellularity and marked distortion of the architecture in the proliferative and hypertrophic zones. Chondrocyte degeneration and death were noted at all levels of the hypertrophic and proliferative zones, suggesting an accelerated disturbance in the life-to-death cycle of the chondrocytes. The current study examines the mechanism responsible for the diminished cell number and whether increased programmed cell death (apoptosis) or necrosis was operative. Proximal femoral growth plates from patients with SCFE (three patients) were prepared and sectioned for histochemistry, in situ detection of apoptosis, and immunohistochemistry. The results showed that the diminished cell number is due to an abnormal frequency and distribution of chondrocytes undergoing apoptosis. Although it is unclear whether the increased apoptosis is occurring early or late in the disease, it is highly likely that it is directly linked to pathogenesis.

Bcl-2 positively regulates Sox9-dependent chondrocyte gene expression by suppressing the MEK-ERK1/2 signaling pathway.

Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.

Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype.

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.

Age-related expression patterns of Bag-1 and Bcl-2 in growth plate and articular chondrocytes.

Aging cartilage displays increased chondrocyte apoptosis and decreased responsiveness of chondrocytes to growth factors. The molecular mechanisms responsible for these changes have not been identified. Bag-1 is a Bcl-2-binding protein that promotes cell survival, interacts with a diverse group of cellular proteins, and may integrate multiple pathways involved in controlling cell survival, growth, and phenotype. Bcl-2 is important for maintaining chondrocyte phenotype and delaying terminal differentiation and apoptosis of chondrocytes. Comparatively little is known about the role of Bag-1 in cartilage. Here we show that both growth plate and articular chondrocytes in the mouse express the Bag-1 protein. In the growth plate, Bag-1 expression is prominent in the late proliferative and prehypertrophic chondrocytes, displaying a pattern similar to what has been reported for Bcl-2. Further, the expression of both Bcl-2 and Bag-1 declines with age in the articular cartilage. Growth assays demonstrate that knocking down Bag-1 expression causes a decrease in growth rate. These results suggest that Bag-1 is involved in the regulation of chondrocyte phenotype and cartilage aging.

The effects of aging on thermal hyperalgesia and tactile-evoked allodynia using two models of peripheral mononeuropathy in the rat.

The effects of aging on the behavioral manifestations of neuropathic and inflammatory pain were investigated using two models of peripheral nerve injury. The left sciatic nerve of young and aged Fischer 344 FBNF1 hybrid rats (4-6 and 24-26 months old, respectively) was ligated using either the chronic constriction injury (CCI) model of Bennett and Xie or the partial sciatic nerve ligation (PSNL) model of Seltzer et al. A plantar analgesic meter was used to assess age-related differences in CCI- or PSNL-induced thermal hyperalgesia, and nerve injury-induced tactile-evoked allodynia was assessed with von Frey filaments. Aged animals subjected to the PSNL procedure developed a more vigorous and longer lasting thermal hyperalgesic response than did aged rats post-CCI. The CCI model incorporates a more prominent peripheral inflammatory component than the PSNL model. These data support the notion that the peripheral inflammatory response is diminished in aged rats.