One of the most commonly experienced symptoms in dentistry and one that is of major concern to the dentist is pain. Since pain is manifested when an environmental change occurs causing injury to responsive tissues, it is often spoken of as a protective mechanism. Pain has been described by a variety of terms: sharp, burning, aching, cramping, dull and throbbing! (Monheim, 1969). Therefore, it is a difficult term to define. Monheim (1969) describes pain as an unpleasant sensation created by a noxious stimulus that is mediated along specific nerve pathways into the central nervous system, where it is interpreted as pain. Local anesthetics are drugs that will temporarily interfere with conduction when absorbed into the nerve. A blockage of the afferent transmission produces analgesia, while interruption results in motor paralysis. Local anesthetic compounds are the most widely used drugs in dentistry and all are synthetic compounds with the exception of cocaine which no longer is used because of its unusual high toxicity. Structural changes in the molecules of these drugs alter the toxicity, potency, diffusibility, profoundness, and duration of anesthetic activity of these compounds. Frequently, as the length of the anesthetic molecule increases, so does its anesthetic activity. Also, structural changes in the molecule may increase toxicity or irritancy without increasing potency. In most cases toxicity tends to rise as potency increases. Monheim (1969) has suggested that ideal local anesthetics should possess the following properties: (1) Its action must be reversible. (2) It should have a low degree of systemic toxicity. (3) It should have a rapid onset and be of sufficient duration to be advantageous. (4) It should have a potency sufficient to give complete anesthesia without using harmful concentrated solutions. (5) It should have sufficient penetrating properties to be effective as a topical anesthetic. (6) It should be relatively free from production of allergic reactions. (7) It should be stable in solution and readily metabolized by the body. (8) It should be either sterile or capable of being sterilized by heat without deterioration. (9) It must be non-irritating to the tissues and produce no secondary local reaction. No local anesthetic in use today fulfills to perfection all of these requirements. Since local anesthetics are the most commonly used drugs in dentistry for routine procedures when anesthesia is desired, it is of particular interest to study the morphologic changes in the muscles that are situated in close proximity to major sensory nerve fibers to the teeth. It is also of interest to question that local anesthetics are non-irritating to the tissues; in fact, they do produce a definite and precise lesion. Another point of interest is to use and apply local anesthetics to produce a definite lesion clinically to aid in the treatment of a common dental syndrome, the myofacial pain dysfunction syndrome formerly known as Costens Syndrome.

The biochemical and physiologic mechanisms responsible for excisional wound contraction have been extensively studied but are not completely understood. Furthermore, the genes that regulate these processes are unknown. As a result, there are limited treatment options available for patients with diseases or conditions characterized by abnormal tissue contraction. In an attempt to improve our understanding of these mechanisms and identify some of the putative regulatory genes, we studied excisional wound healing in the mid-gestational fetal rabbit. These wounds normally do not contract nor are they associated with inflammation, fibrosis, or scar formation. However, we have been able to induce contraction, with and without inflammation and fibrosis, by providing sustained levels of exogenous TGF-βl and -β3, respectively, at the excisional wound site. In addition to learning more about the role of the TGF-β isoforms in tissue repair, we utilized our model to identify some of the regulatory genes responsible for contraction. We theorized that homeobox genes direct the mechanisms of excisional wound contraction. These genes encode for a family of transcription factors that are known to regulate cellular differentiation and tissue migration during development. We demonstrated that homeobox genes are expressed in normal fetal and adult rabbit skin and their pattern of expression varies for several of these genes. There was a 2-fold increase for Hoxa-5, yet a moderate decrease in Hoxc-6 transcripts in the contracting versus non-contracting fetal group. In addition, there was a greater than 3-fold increase in Hoxd-8 transcripts in the adult excisional wound group compared to the normal adult skin group. Using a ribonuclease protection assay, we confirmed that the level of Hoxd-8 expression was significantly greater at the site of adult excisional wounds than in the normal skin. These findings suggest that this homeobox gene may regulate some aspect of the mechanisms responsible for excisional wound contraction. Further studies may delineate the role of Hoxa-8 and other homeobox genes in the mechanisms of excisional wound contraction. If they are determined to direct various aspects of the contractile process, future therapeutic interventions targeting these genes may improve the healing of many abnormal wound healing conditions and diseases.

The spermatozoon has been the subject of numerous investigations because of its remarkable specialization for locomotion and fertilization. Although the spermatozoon of the guinea pig is relatively large when compared with other mammalian spermatozoa, its small size has hidden many of the details of its structure from the light microscopist. It was not until the invention of the electron microscope that a thorough study of spermatozoon structure could be accomplished. The following anatomical description is based on the works of Fawcett and Hollenberg (1963), Fawcett (1965), and Fawcett (1970). The principle parts of the guinea pig spermatozoon are the head and the tail with the tail being subdivided into the neck, middle-piece, principal-piece, and the end-piece. These and other anatomical structures which will be discussed are depicted in figures I and II which were taken from the work by Fawcett (1965).

There are no data available as to whether training with eccentric contractions are more effective than concentric contractions for producing skeletal muscle fiber hypertrophy. To better understand the effects of training with different contraction types two related studies were performed. In the first study a device which is frequently used by clinicians for concentric and eccentric exercise, the Kin-ComR, was tested for accuracy and reliability. The measurements obtained from the force, angle, and speed transducers of this device were found to be accurate and reliable between days. The purpose of the second study was to determine if there was a difference in the percent change of fiber area in the vastus lateralis muscle as a result of concentric and eccentric exercise at equal power levels. Twenty normal subjects were randomly assigned to two groups. Both groups exercised three times per week for four weeks on the Kin-Com dynamometer. One group performed concentric contractions of their right quadriceps femoris muscle at an intensity of 90% of their maximal concentric power through a range of 75° of knee extension. The other group performed eccentric contractions at the same relative power level. Needle muscle biopsies were obtained from the vastus lateralis muscle before and after the exercise program. Muscle fiber type differentiation was performed using a myosin adenosine triphosphatase stain at an alkaline preincubation. The percent change in fiber area was determined for each fiber type for each subject and a one-way ANOVA was used to analyze the data. Our results showed that the type II fibers of the concentric group exhibited a greater percent increase in area as compared to the eccentric group. The percent change in isometric torque was determined for each subject and a one-way ANOVA was performed on the data. The results showed that the concentric group increased maximal isometric torque production more than the eccentric group. Our results indicate that when exercising at the same relative power level a subject performing concentric contractions will 1) show greater muscle hypertrophy and, 2) improve in isometric torque production more than a subject training with eccentric contractions.

Primary brain tumor affects some 18,000 adults in the United States each year (Silverberg et al., 1990; Merchant et al., 1997) and over 30% are high-grade anaplastic astrocytoma or glioblastoma multiforme (GBM) (Parney et al., 1997). According to Kruse et al., 1989, the treatment of patients with recurrent or persistent high-grade gliomas represents a major therapeutic challenge. The use of conventional therapy consisting of surgery, followed by radiation therapy and chemotherapy for gliomas, has been relatively ineffective (Jaecle et al., 1994) despite the fact that these therapies are cytoreductive in nature (Black, 1991). Most malignancies will recur locally but may also reappear at a different site within the brain. A brain tumor, once established, usually continues to outstrip the inhibitory action of any immunobiological defense mechanisms against it. Thus, malignant intracranial (IC) brain tumors represent a lethal neoplastic disease in which treatment has failed to extend the lifespan of afllicted individuals, with a GBM having a median survival rate of less than one year (Harsh et al., 1987).This has prompted a search for other potentially useful methods to better understand the biology of brain tumors as well as better ways to treat them The studies outlined herein, addressed the mechanisms behind the protection against tumor development provided by the various cells of the innate and cellular immune response in a rat brain tumor model. Investigations consisted of: 1) primary (1°) vaccination involving a phenotypic examination and functional analysis of the cells of the innate immune response and the cells of the adaptive immune response infiltrating an RT-2 glioma and the expression of the CD25 receptor; 2) 1° and secondary (2°) vaccination that involved a follow-up on survival as well as the phenotyping of cells of the leukocytic immune response; 3) rechallenge of long-lived rats, from Experiment 2, in the contralateral (left) hemisphere; and 4) acquisition of memory T lymphocytes from vaccinated rats and the use of lymphocyte depletion studies to determine which cells were necessary to provide a protective vaccination against development of tumor. Thus, this study illustrated a potential therapeutic strategy to develop treatments for GBM patients as well as providing protection against development of tumor by the use of vaccines.

Precise connections in many mammalian nervous systems require a great deal of remodeling during development. In the visual system, many excess synapses are originally formed in the lateral geniculate nucleus and striate cortex. Only the correct set of axon terminals are retained during normal development, while imprecise ones withdraw. The mechanism by which only correct axons are retained requires neural activity, and may be regulated by specific receptors at synapses. The transmission of neural signals at these synapses is carried out in part by the glutamate-activated NMDA receptor. It is hypothesized that NMDA receptor activation plays a crucial role in enhancing only those connections in the immature system which will form a retinotopically correct map in the LGN and cortex. NMDA receptor activation requires depolarization of the neuron membrane. Possibly, only neurons transmitting information from nearby areas in the retina summate to produce NMDA receptor- mediated currents. The result is an influx of Ca++ ions that has been shown to cause trophic effects within the cell that could enhance the synaptic connection. Thus, NMDA receptors may act to detect coincident neural activity in immature animals, thereby allowing only visuo-topically related axon terminals to undergo enhancement of synaptic transmission and structure. As development proceeds, NMDA receptor function decreases, possibly reducing these intracellular effects. Blocking NMDA receptor activation experimentally does alter the normal set of connections in the visual system. Yet, is there a direct cause- and-effect relation between NMDA receptor activity and anatomical changes? Many cellular events probably result from NMDA-mediated currents. Intracellular changes in phosphorylation states and protein levels could eventually alter a synapse at the anatomical level. Study of the changing NMDA receptor subunit types making up the receptor within visual system structures could reveal, in part, the means by which plasticity is down-regulated. The experimental regulation of these subunits in vivo could reveal important information concerning their specific function if plasticity and development were to be altered as a result. A summary of previous studies, and proposals for further research concerning the role of the NMDA receptor and its various types in developing visual pathways are presented in this manuscript.

Previous studies have determined adenovirus mediated herpes simplex thymidine kinase gene transfer (AV-TK) to be effective for the treatment of experimental gliomas. In this study we report three distinct phenomenon. First, animals with complete regression of subcutaneous tumors upon intratumoral injections of AV-TK with concomitant Ganciclovir® (GCV) administration developed tumor immunity. These animals had the ability to reject a subsequent inoculum of lethal doses of tumor cells. This tumor immunity was long standing and protective as far as 6 months from the time of initial tumor ablation. Of interest, adoptive transfer of splenocytes from AV-TK treated-tumor ablated animals to naive animals conferred resistance to tumor formation upon injection of lethal doses of tumor cells. This data strongly indicated the mechanism of tumor immunity was cell mediated. Further analysis of the anti-tumor immune response implicated CD8a83 cytotoxic T-lymphocytes as the effector cell. Animals with complete tumor regression survived over 300 days and showed no signs of tumor relapse. Therefore, treatment of solid unifocal tumors with AV-TK and GCV may be able to prevent tumor relapse through the generation of an anti-tumor immune response. Secondly, we determined that AV-TK and GCV treatment efficacy was dependent on tumor antigenicity. Two different subcutaneous tumor models were utilized; the weakly immunogenic 9L and the strongly immunogenic RT2. For the same dose of intratumorally injected AV-TK, a greater percentage of RT2 tumor were eliminated as compared to 9L. Final survival efficacy was dependant on the tumor type and the initial tumor size. In studying the importance of host immunity in tumor progression, we have determined that in vivo, the GCV mediated bystander effect was not sufficient to result in tumor eradication without involvement of a host immune response. In athymic rats, 9L tumors failed to regress upon AV-TK and GCV treatment. In contrast, tumors of similar size were ablated upon treatment in immunocompetent animals. Of related significance, adenovirus mediated gene transfer facilitated generation of tumor immunity. Animals with tumors ablated by AV-TK and GCV treatment developed an anti-tumor immune response which was protective against further tumor engraftment. In contrast, alternative treatments such as surgical excision of subcutaneous gliomas or tumor vaccination was not sufficient to protect against secondary tumor challenge. Injection of adenovirus altered the amount and phenotypes of tumor infiltrating lymphocytes from the NK phenotype towards tumor specific CD8+ CTL cells. This immunomodulatory property was potentially responsible for generation of the immune response. Therefore, AV-TK was effective through two mechanisms. Transfer of the HSVTK gene conferred GCV sensitivity resulting in substantial tumor regression and the adenovirus backbone served as an immune adjuvant to augment generation of host tumor immunity. This immunomodulatory property of adenovirus vectors is an added advantage to their use for cancer gene therapy.

Nine lateral rectus motoneurons of the principal abducens nucleus, intracellularly stained with HRP, were morphometrically analyzed by light microscopy using a new method for determining motoneuron size. Particular emphasis was placed on devising a method of estimating total dendrite size from the proximal dendritic diameter alone. The dendrites of these cells were divided into three types. One type, the microdendrites, had a consistent diameter of l micrometer, variable but short lengths, and added very little to the overall cell size. The majority of the dendrites on these cells (83) were standard in appearance but they could be separated into two further types. Six dendrites differed from the other 77 in that they were tapering processes which branched minimally, had both a rostrally and a caudally directed secondary dendrite and showed a larger ratio for the sum of the secondary dendrite diameters to the proximal dendrite diameter. The remaining 77 branched extensively and traveled either rostral or caudal in the brainstem. However, the most significant difference was quantitative. The tapering dendrites were approximately 2X the size of the prevalent branching dendrites based on proximal diameter measurements. Correlation coefficients of the relation between proximal diameter and surface area or volume of the entire dendrite increased when the correlations were separated into two types. Therefore, to insure the most accurate total size calculations, the regression lines used for estimating dendrite size were of the separate correlations. Total neuron size was calculated by adding the soma and dendrite surface areas. An intraneuronal comparison of size indicated that the size of the soma was not indicative of the size of the cell and it constituted between 2% to 7% of the total cell size. Comparison of the motoneuron size to the mechanical properties of their muscle units was inconclusive. However, a general tendency for small motoneurons to innervate muscle units of lower force output was observed. The smaller motoneurons were generally more dorsally located in the nucleus.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.