Denise McBurney

Microglial proliferation in the spinal cord of aged rats with a sciatic nerve injury

Nerve injury may lead to chronic neuropathic pain syndromes. We determined whether the extent of central nervous system microglial activation that accompanies nerve injury is age dependent and correlated with behavioral manifestations of pain. We used the Bennett and Xie sciatic nerve chronic constriction injury model (Bennett, G.J., Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1998) 87-107) to induce neuropathic pain in three age cohorts of Fischer 344 FBNF1 hybrid rats (4-6, 14-16, and 24-26 months). Rats were assessed for thermal sensitivity (hyperalgesia) of their hind paws pre-injury (day 0) and up to 35 days post injury. On various days post injury, the L4-L5 levels of their spinal cords were reacted for localization of an antibody to OX-42, a marker for microlgia. OX-42 immunoreactivity (ir) was quantified by use of a Bioquant density analysis system. OX-42 ir was heavy in areas of sciatic nerve primary afferent terminations and in the motor columns of its neurons. Aging increases OX-42 ir in the absence of injury. After injury, OX-42 ir increased further, but the increases over control levels decreased with age. Ligation-induced analgesia and hyperalgesia were both correlated with the increases in OX-42 ir, regardless of age.

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 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.

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 Cα and NFκB 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.

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.

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. J. Cell. Biochem. 102: 786–800, 2007.

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.

Phentermine+fenfluramine produce cocaine-like discriminative cues.

Drug discrimination studies were conducted in six male Sprague-Dawley rats trained to discriminate the interoceptive cues produced by 10 mg/kg cocaine in an effort to investigate if there is stimulus generalization to phentermine or phentermine + fenfluramine. Once having reached criterion performance, these rats were tested with lower doses of cocaine and generated a typical dose-response curve allowing for calculation of an ED50 value: 2.798 mg/kg. Testing of phentermine in doses of 1.25-5.0 mg/kg indicated generalization with the highest dose producing 80% cocaine-appropriate responding and allowing for an ED50 value of 2.356 mg/kg. When the phentermine doses were tested in combination 2.0 mg/kg fenfluramine, however, there was an increase in the discriminability of the highest phentermine dose and a slight decrease in the ED50 value of the combination. Thus, administration of phentermine + fenfluramine, having both dopamine-releasing and serotonin-releasing properties, respectively, may mimic the neurochemical activity by which cocaine acts in the central nervous system and may possibly allow for cocaine-like effects as these two drugs see increased use in obesity control.