Multiple complex somatosensory systems in mature rat molars defined by immunohistochemistry.

OBJECTIVE: Intradental sensory receptors trigger painful sensations and unperceived mechanosensitivity, but the receptor bases for those functions are only partly defined. We present new evidence here concerning complex endings of myelinated axons in rat molars. DESIGN: We sectioned mature rat jaws in sagittal and transverse planes to analyze neural immunoreactivity (IR) for parvalbumin, peripherin, neurofilament protein, neurotrophin receptors, synaptophysin, calcitonin gene-related peptide (CGRP), or mas-related g-protein-receptor-d (Mrgprd). RESULTS: We found two complex sensory systems in mature rat molar dentin that labeled with neurofilament protein-IR, plus either parvalbumin-IR or peripherin-IR. The parvalbumin-IR system made extensively branched, beaded endings focused into dentin throughout each pulp horn. The peripherin-IR system primarily made unbeaded, fork-shaped dentinal endings scattered throughout crown including cervical regions. Both of these systems differed from neuropeptide CGRP-IR. In molar pulp we found peripherin- and parvalbumin-IR layered endings, either near special horizontal plexus arrays or in small coiled endings near tangled plexus, each with specific foci for specific pulp horns. Parvalbumin-IR nerve fibers had Aß axons (5-7µm diameter), while peripherin-IR axons were thinner Aδ size (2-5µm). Mechano-nociceptive Mrgprd-IR was only found in peripherin-IR axons. CONCLUSIONS: Complex somatosensory receptors in rat molars include two types of dentinal endings that both differ from CGRP-IR endings, and at least two newly defined types of pulpal endings. The PV-IR neurons with their widely branched, synaptophysin-rich, intradentinal beaded endings are good candidates for endodontic non-nociceptive, low threshold, unperceived mechanoreceptors. The complex molar dentinal and pulpal sensory systems were not found in rat incisors.

Gene expression analysis characterizes antemortem stress and has implications for establishing cause of death.

Within the field of forensic pathology, determination of the cause of death depends upon identifying physical changes in the corpse or finding diagnostic laboratory abnormalities. When such perturbations are absent, definitive assignment of a cause of death may be difficult or impossible. An example of such a problem is sudden infant death syndrome (SIDS), a common cause of neonatal mortality that does not produce physical findings or laboratory abnormalities. Although respiratory failure as a cause of SIDS represents the most widely held hypothesis, sudden cardiac death and hyperthermia have also been advanced as possible causes. We hypothesize that each of these physiological stresses would produce a different pattern of premortem gene expression and that these patterns of gene expression would remain evident in tissues collected postmortem. If these patterns were sufficiently distinctive, they could be used to identify the cause of death. Using an infant mouse model, we compared gene expression patterns in liver tissue after sudden death, lethal hyperthermia, and lethal hypoxia. Each of these conditions produced readily distinguishable differences in gene expression patterns. With the K-nearest neighbor classification algorithm, only 10 genes are necessary to correctly classify samples. If the liver tissue was not harvested immediately after death, additional alteration in gene expression patterns resulted; however, these alterations did not affect the group of genes used to classify the samples. Our findings suggest that gene expression analysis from tissues collected postmortem may provide useful clues about certain physiologic stresses that may precede death.

Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos.

BACKGROUND: Using vital dyes, we have previously shown that while hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death within specific tissues (e.g., neuroepithelium) of day 9 mouse embryos, cells of the heart are resistant to the cell death-inducing potential of these teratogens. Subsequent work has shown that teratogen-induced cell death is associated with activation of the mitochondrial apoptotic pathway, i.e., release of cytochrome c from mitochondria, activation/cleavage of procaspase-9, -3, and -2, inactivation of poly(ADP-ribose) polymerase, and internucleosomal fragmentation of DNA, whereas resistance to teratogen-induced cell death in the heart is associated with a failure to activate this pathway. Teratogen-induced activation of the mitochondrial apoptotic pathway is initiated between 2.5 and 5 hr after teratogens are added to the culture medium. Because both the heart and the surrounding yolk sac are essential to successful development of mouse embryos during early postimplantation mouse development, we hypothesized that cells of the yolk sac are also resistant to teratogen-induced cell death. METHODS: To test our hypothesis, we cultured day 8.5 mouse conceptuses (embryo plus yolk sac) in whole embryo culture. On the morning of day 9, conceptuses were exposed to HS (43 degrees C for 15 min and then returned to 37 degrees C), 4CP (40 microM, 5-10 hr), or ST (0.5 microM 5-10 hr). At 5 and 10 hr after addition of teratogen, conceptuses were removed from culture and dissected into embryo and yolk sac. Activation of the mitochondrial apoptotic pathway was then assessed separately in embryos and yolk sacs using Western blot analysis to detect activation of procaspase-9, -3, and -2, enzyme assays to measure caspase-3-like activity, and immunohistochemistry to detect caspase-3 activation/cleavage in yolk sac cells. RESULTS: Although Western blot analysis revealed that procaspase-9, -3, and -2 were activated/cleaved in the embryo as early as the 5-hr time point, activation/cleavage of these caspases could not be detected in the yolk sac at either the 5- or 10-hr time point. Using an enzyme assay, we determined that caspase-3-like activity in the yolk sac was induced 1.7-fold by HS, 4.4-fold by 4CP, and 3.3-fold by ST. This compares to the embryo in which caspase-3-like activity was induced 45-fold by HS, 26-fold by 4CP, and 45-fold by ST. Using an antibody specific for the active p17 subunit of caspase-3 and immunohistochemistry, we were able to detect a small number of yolk sac cells showing caspase-3 activation. Thus, the low-level induction of caspase-3-like activity in the yolk sac is in part related to activation/cleavage of procaspase-3. CONCLUSIONS: Results presented indicate that cells of the extraembryonic yolk sac, like cells of the embryonic heart, are substantially more resistant to teratogen-induced activation of the mitochondrial apoptotic pathway and subsequent apoptosis compared to other embryonic tissues, particularly cells of the neuroepithelium.