Date of Award

1972

Document Type

Senior Scholars Paper (Open Access)

Department

Colby College. Biology Dept.

Advisor(s)

Easton, W. T.

Second Advisor

George S. Maier

Abstract

It has been clearly demonstrated that the barbels of Ameiurus nebulosus regenerate completely following amputation. The current investigations involve experimentation dealing primarily with determination of the mechanisms by which regeneration occurs. However, prior to conducting a series of experiments designed to elucidate the processes which initiate regeneration, several preliminary investigations to determine normal barbel structure and regeneration were made.

Brain dissection of an adult catfish confirmed that innervation of the barbels is by both the fifth and seventh cranial nerves, and not solely by the seventh cranial nerve as suggested by Olmstead (1920). Further information regarding normal barbel innervation and structure was obtained from an histological examination of longitudinal and cross sections. The barbel is supported by a central cartilaginous axis, which is surrounded by a perichondrium. Enclosing the perichondrium is a layer of looser connective tissue, containing nerve fibers concentrated chiefly dorsal and ventral to the cartilage, and an artery. Interspersed among the squamous and cuboidal epithelial cells which cover the barbel are numerous taste buds and pigment calls. Because taste buds of two distinct shapes were observed, the possibility that there may be two types of sensory buds in the barbel cannot be overlooked. One undoubtedly serves the function of teste, while the other may be used for touch.

Observation of the histological details of normal regeneration indicates that the wounded stub of an amputated barbel is healed within one day by a layer of squamous epithelial cells. Despite rapid healing, elongation of the regenerate does not commence until a blastema is formed, apparently from migrating perichondrial cells which deplete the perichondrium proximal to the amputation level. As the blastema cells undergo rapid proximo-diatal proliferation and the epidermal cells continue mitotic divisions, the barbel enters a period of rapid elongation. At 20°C regeneration proceeds at a rate of 1.0-1.5 mm. per week until the original dimensions have been regained. Regeneration is inhibited by colder temperatures, and 1s totally halted at 12°C. Because nerves, blood vessels, and connective tissue are not fully regenerated until the original length has been approximated, the regenerating barbel diameter is somewhat smaller than that of the original.

To substantiate the conclusions drawn by histological observation of normal regenerating barbels, the barbels of many fish were subjected to abnormal treatments. The first treatment involved extirpation of the cartilaginous rod prior to amputation of the barbel in the affected region. The subsequent regeneration is abnormal and inhibited. In the absence of the cartilaginous rod and its surrounding perichondrium, no blastema forms and therefore little elongation occurs due to lack of physical pressure apparently exerted on the wound-healing epidermal and connective tissue cells by the proliferating and chondrifying blastema cells. Even without blastema formation, nerves, connective tissue, blood vessels, and epidermis, with its pigment cells and taste buds, regenerate. Sub-dermal and dermal connective tissue rapidly grow beneath the healed epidermis, but do not form a zone similar to, or part of, the blastema. Because the blastema does not develop without the presence of the cartilage and perichondrium, it appears that the blastema is derived primarily from perichondrial cells and to a limited extent from chondrocytes released from the severed cartilage. It further appears from observation of normally regenerating barbels that the blastema is initially derived from migrating rather than proliferating perichondrial cells.

To determine if the blastem originates from migrating or dividing perichondrial cells, the fish were subjected to a second abnormal treatment. Regenerating barbels were injected with either mitomycin-C or aza-guanine to inhibit DNA synthesis, and thereby also to inhibit mitotic cell division. Although it was not possible to totally inhibit cell division without killing the fish, mitosis was significantly inhibited in many cases. Following antibiotic injection, the wound was healed as usual, but by an abnormally thin layer of epithelial cells. Although a few subdermal connective tissue cells penetrated between the cartilaginous tip and the wound epithelium, most of the cells were prevented from penetrating beneath the wound epithelium, and therefore accumulated proximal to the amputation point. Because the antibiotics apparently inhibited mitosis

sufficiently to prevent formation of the space necessary to allow perichondrial cells to accumulate at the severed tip, the perichondrial cells also were sterically hindered from penetrating between the epidermis and the cartilage. However, unlike the case of the connective tissue cells where there was an abnormally large accumulation of cells immediately proximal to the level of amputation, there was no abnormal cell accumulation in the case of the perichondrial cells, indicating that they had not undergone even inhibited mitosis. It therefore appears that the perichondrial cells form the blastema by migration to the severed cartilaginous tip, rather than by proliferation.

Further evidence in support of the hypothesis that the blastema is derived from migrating perichondrial cella was sought by a third treatment. Many fish were injected with various dosages of the vital dye trypan blue, which is specific for phagocytic cells. Intraventricular injection was unsuccessful because the fish died shortly after the injection, while intraperitoneal injection was also somewhat unsuccessful because only very small quantities of the dye reached even the base of the barbels, and almost none extended significantly into the barbels. Therefore, the destiny of the colored cells following amputation could not be traced because there were no colored cells as far distal as the excisions. Because it is felt that the basis for the trypan blue experiments is sound and that with improved injection

technique, the experiment would be successful, the author intends to pursue the problem further.

A fourth treatment, removal of barbel epidermis, was used to determine the origin of new epithelial cells. As an adaptive reaction to frequent natural abrasions in the epidermis, epidermal cells are capable of rapid proliferation, resulting in wound healing, by spreading over or into a wound area from its margins.

Keywords

Regeneration (Biology), Catfishes

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