Tritiated Thymidine Labeling Research Articles

Effects of sodium cholate on experimental carcinogenesis and cell proliferation in an excluded colonic segment.

Bile salts appear to be important promoters of colon carcinogenesis. This study was designed to assess the importance of the fecal stream in cholic acid-induced colon tumor promotion. Male Sprague-Dawley rats underwent transverse colostomy after induction with dimethylhydrazine (DMH) and the excluded distal colon was irrigated with saline or sodium cholate (23 microM) 5 times per week until sacrifice. Controls initially injected with saline were similarly treated. All surviving animals were sacrificed at 28 weeks after the last DMH injection. Five animals from each group were randomly chosen to assess tritiated thymidine labeling and distribution by autoradiography in normal appearing colon mucosa of irrigated bowel. Cholate irrigation failed to increase tumor yield or modify the proportion of adenomas and adenocarcinomas in this model. Paradoxically, fewer tumors per affected rat were noted with sodium cholate irrigation. Cholate irrigation also failed to affect crypt cellularity, thymidine labeling indices, and labeling distribution in DMH-treated rats and controls. An effect of DMH was seen, however, with an increase in thymidine labeling index and increased labeling in the top half of the crypt in all DMH-treated groups. This study suggests that tumor promotion with primary bile salts is not a direct affect and may result from further bile salt metabolism within the fecal stream. DMH-induced changes in cell proliferation were reproduced with this model. Use of an excluded colon segment to assess the effect of suspected tumor promoters on carcinogenesis or colon mucosal cell proliferation is feasible and may be a useful model for future studies.

Tritiated-thymidine labeling of rectal epithelial cells in ‘non-prep’ biopsies of individuals at increased risk for colonic neoplasia

Recent measurements have shown increased proliferation of colonic epithelial cells in individuals at heightened risk for cancer of the large intestine. This biomarker has facilitated measurements of the effects of nutritional intervention in studies that are attempting to inhibit tumor development in high-risk individuals. In this study, further measurements were made of the proliferation of rectal epithelial cells, when biopsies were removed from mucosa that had not previously been disturbed by any tapwater or other enema preparations. Progressive increases were found in the numbers of [ 3H]dThd-labeled epithelial cells in rectal crypts, and in labeling index profiles, in patients having previous sporadic adenomas or colon cancer, compared to individuals who had not developed colonic neoplasms. The most quiescent proliferative equilibrium was found in individuals without previous colonic disease. Findings indicated that ‘non-prep’ rectal biopsies obtained from the most accessible region of the large intestine, show modifications in the biomarker of cell proliferation paralleling colon cancer risk.

Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas.

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI). Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study. In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.

Hepatic adenoma within a spindle cell carcinoma in a woman with a long history of oral contraceptives

The case of a young woman is presented who had a long history of oral contraceptive use and who was found to have a focal hepatic adenoma within a large spindle cell carcinoma of the liver. The pleomorphic tumor is positive for keratin and alpha 1-antitrypsin by immunohistochemistry, has intracellular lumina by electron microscopy, a moderately high thymidine labeling index, and is negative for estrogen and progesterone receptors. A review of the literature concerning liver tumors occurring in young patients indicates that there is an underlying disturbance of the hormonal milieu in all instances reported. The prognosis is widely variable and unpredictable.

Centripetal spread of arterial collateral endothelial cell hyperplasia after renal artery stenosis in the rat.

The factors responsible for collateral arterial growth after major artery occlusion remain obscure, despite their importance for tissue survival. An increase in endothelial cell labelling with tritiated thymidine, as an index of collateral arterial growth, occurs early after renal artery occlusion. Our working premise was that an increase in endothelial cell labelling would occur simultaneously throughout the length of the collateral arteries if biophysical factors related to blood flow were the responsible mechanism, because blood flow must be increased simultaneously throughout the length of the small, preformed collateral arterial vessels. On the other hand, if the information spread from the ischemic zone, one would anticipate centripetal spread of the endothelial cell hyperplasia in a retrograde direction from the ischemic zone. With the periureteric collateral arterial supply as the model, we performed serial studies of tritiated thymidine labelling following renal artery stenosis in the rat. As anticipated, endothelial cell labelling rose sharply within 24 to 48 hours, first evident in the area immediately adjacent to the renal hilum. Thereafter, a progressive, time-related centripetal gradient in endothelial cell tritiated thymidine labelling occurred (p less than 0.01). These findings indicate that the factors responsible for endothelial cell hyperplasia are less related to blood flow in the lumen than to downstream, ischemic events. Although the mechanism responsible for the centripetal spread remains speculative, the communication system is likely to involve cell-to-cell contact in the vessel wall.

Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: Implication for trophoblast proliferation and differentiation

Cytologic localization of epidermal growth factor receptor and myc oncogene protein product in developing human placenta was analyzed by avidin/biotin immunoperoxidase techniques with a monoclonal antibody to epidermal growth factor receptor and an affinity purified polyclonal antibody to myc protein product. Epidermal growth factor receptor was found to be almost exclusively localized to syncytiotrophoblast, paralleling the immunohistochemical localization of human chorionic gonadotropin. Since epidermal growth factor has been shown to stimulate human chorionic gonadotropin and human placental lactogen production by cultured early placental tissue, the present finding that epidermal growth factor receptor was localized to mitotically inactive syncytiotrophoblastsuggests a role for epidermal growth factor receptor in the induction of differentiated function of trophoblast rather than trophoblast proliferation. By contrast, myc protein product was found to be predominantly localized to cytotrophoblastic cells, paralleling the autoradiographic distribution of replicating cytotrophoblast identified by tritiated thymidine labeling of placental explant. A close similarity between the cytologic localization of myc protein product and tritiated thymidine labeling of placental explant suggests that myc protein expression is linked to trophoblast proliferation. Furthermore, immunohistochemical cellular levels of both epidermal growth factor receptor and myc protein product were most pronounced in early placenta and declined in term placenta. Thus myc protein product and epidermal growth factor receptor seem to play a crucial role in the induction of trophoblast proliferation and differentiation, respectively, during the development of human placenta.

Tritiated thymidine labeling index in indian childhood acute lymphoblastic leukemia

Tritiated thymidine labeling index (LI) was determined in 25 unselected children with cytochemically proved ALL, at various stages of the disease. All the cases were treated by a uniform therapeutic protocol. Pretreatment LI determined in 15 cases showed an inverse correlation with the duration of first complete remission. LI was also studied during remission in 23 patients. Six out of these 23 patients relapsed within subsequent 90 days, while the rest continued to be in remission. Mean LI of the nonerythroid cells in later group of patients was found to be significantly higher than that of the patients who relapsed.

Influence of the prostaglandin E1 analogue Rioprostil on the human gastric mucosa.

Ten healthy volunteers received the prostaglandin E1 analogue Rioprostil 300 micrograms b.i.d. for 1 week. Endoscopically obtained biopsies were investigated with tritiated thymidine and autoradiography to determine the rate of cell proliferation and with a texture-analyzing system to measure the intracellular amount of mucus in the epithelial cells. Rioprostil did not alter the tritiated thymidine labeling index in the antral area but did significantly depress it in the fundic area. The mucus content was unchanged in the antrum but was significantly increased in the fundus. These observations indicate that Rioprostil given orally causes an enhanced cell maturation in the fundic area as well as an increased intracellular mucus content. Rioprostil seems to have no influence on the human antral area.

Label-Retaining Cells in Human Embryonic and Fetal Epidermis

Human embryonic and fetal epidermis was examined and labeling indices (LIs) for basal, intermediate, and periderm cells were determined. The LI for fetal basal cells was 8-11% and the LI for fetal intermediate cells was 7.5-9%. The total fetal epidermal LI was 16-20%, which equaled the basal LI for embryonic epidermis. After 21 days in organ culture, only basal cells in the fetal epidermis labeled with tritiated thymidine, while both basal and intermediate cells in the embryonic epidermis labeled and the total LI for fetal and embryonic epidermal cells was the same as the adult epidermal LI (7%). The LI for periderm decreased with increasing estimated gestational age (EGA) from 9.5% at 49 days EGA to 0.4% at 85 days EGA. A subpopulation of epithelial cells that retained tritiated thymidine label and that have some of the attributes associated with stem cells has been previously demonstrated in rodents. In order to examine human embryonic and fetal epidermis for the presence of such cells, epidermis from various gestational ages were labeled and grown in organ culture for 21 days. The mean percent label-retaining cells (LRCs) for embryonic and fetal epidermis was determined. Approximately 4% of the embryonic and 2% of the fetal epidermal cells retained label for 21 days in organ culture. Embryonic LRCs were found in the basal and suprabasal layers, but fetal LRCs were found only in the basal layer. The presence of LRCs in human embryonic and fetal epidermis suggests that epithelial cell proliferation in these tissues may be regulated via a stem cell pattern of proliferation.

High-degree abnormalities of nuclear DNA distribution in SIg negative acute lymphoblastic leukemia with vacuolated blasts

Among 65 consecutive patients with untreated adult acute lymphoblastic leukemia (ALL), six had peculiar clinical and cytologic features. Bone marrow and circulating blasts were large and showed prominent vacuolization, with variable degree of granular periodic acid-Schiff positivity in the cytoplasm. All patients had marked spleen and liver enlargement with cholestasis and without either lymph node or central nervous system disease. They achieved complete response to chemotherapy, and their median survival was longer than 12 months. Blast cells were studied for immunologic markers, propidium iodide flow cytofluorometric DNA content, and in vitro tritiated thymidine labeling index (3H-TdR LI). These showed a "null" phenotype in two cases, contained intracytoplasmic immunoglobulins (Cy mu) in two and were positive for CALL and Ia antigens in two others (one of which also contained Cy mu). Surface immunoglobulins (SIg) were found on less than 12% of cells, and T-cell markers were absent. In five cases, one or two stem lines with highly abnormal modal DNA content were found to coexist with diploid blasts. The 3H-TdR LI was high (8.5%-15.5%). Abnormal cell lines, after being cleared by chemotherapy, were found again at relapse. Additional cell lines appeared during the course of the disease in one patient, having different sensitivity to cytostatics. These cases of ALL apparently derive from B-cells not yet expressing SIg and have an exceptionally high incidence of aneuploid cell lines.

Intimal cell mass-derived atherosclerotic lesions in the abdominal aorta of hyperlipidemic swine: Part 2. Investigation of endothelial cell changes and leukocyte adherence associated with early smooth muscle cell proliferative activity

We have investigated several aspects of endothelial cell (EC) behavior during the initiation and early development of intimal cell mass (ICM)-derived atherosclerotic lesions in the distal abdominal aortas of young swine fed hyperlipidemic (HL) diets for 0, 14, 49, or 90 days. By scanning electron microscopy no breaks in endothelial integrity or other abnormalities were observed even at 90 days on diet after lesions were well established. Also, counts of leukocytes adherent to the endothelium by both scanning and light microscopy revealed no greater numbers in HL than in mash control swine. Estimates of individual EC losses over ICM-lesions in the HL swine (based on calculations from tritiated thymidine labeling indices and EC growth rates determined by counts) suggested a loss of approximately one per 100 EC/day. This loss over ICM lesion was not significantly greater than that over ICM in mash controls, but was significantly greater than that of EC not over ICM lesions in the same HL animal. In any event, the estimated loss seems to be too trivial for the endothelial barrier to be compromised even transiently in a biologically significant fashion. A significant correlation was observed between tritiated thymidine labeling indices of cells within the ICM lesions and of those of the overlying EC that was not observed with the ICM in the controls. Possibly the positive correlation may be a result of the abluminal surface of the overlying EC being exposed to the abnormal milieu of the ICM lesion. It is emphasized that results reported here apply only to ICM-derived lesions at an early stage of development and that they do not contradict results obtained by others with other lesion types such as those derived from monocytes. Furthermore, in later stages of development of ICM-derived lesions in the same model we know that extensive endothelial cell damage can be demonstrated. Also, functional changes in endothelial permeability may have been present in early stages that would not have been detected with the methods used in this study.

Cancer family syndrome: Marker studies

Individuals from kindreds with the cancer family syndrome (CFS) have an increased hereditary risk for the development of adenocarcinoma of the colon in childhood and early adulthood. Previous studies have suggested that this high occurrence of adenocarcinoma may be due to a genetic defect in the control of colonic epithelial proliferation. Others have suggested that these families may have an underlying abnormality in immunologic tumor surveillance. We have investigated these possibilities in 15 cancer-free, at-risk individuals (10 children, ages 3–15 yr, and 5 adults) from two unrelated CFS kindreds. Colonic mucosal proliferative activity was studied by in vitro autoradiography after tritiated thymidine labeling in 7 subjects. The mean labeling index (12.7 ± 0.9%) was comparable to that in controls, as was the distribution of thymidine labeling. Immunologic evaluation revealed depressed lymphocyte culture responses to stimulation by microbial antigens, but not to that by mitogens. Mixed lymphocyte culture responses were depressed in 4 of 8 subjects, but became normal in 2 of these after filtration through a Sephadex G10 column. Natural killer cell cytotoxicity was significantly depressed in 5 of 13 subjects, and borderline normal in another 3 subjects. These data suggest that many cancer-free members of CFS kindreds have a spectrum of in vitro cell-mediated immunologic defects that might interfere in vivo with the recognition or killing of incipient tumor cells.

Tritiated thymidine labelling in Vitro of human cancer of the breast: Counting error and sampling error

Tritiated thymidine labelling indices (TLIs) were determined on a number of primary cancers of the human breast. Twenty-two slides were chosen which demonstrated a wide range of TLIs and each was counted twice to assess ‘counting error’. TLIs derived from successive counts of the same slide showed a coefficient of variance greater than 25% in half of the slides, but a significant difference between the two ( P < 0.05) in only 2 of 22 pairs. When TLIs derived from paired specimens taken from different sites in each of 22 tumours were compared, there was found to be a significant difference ( P < 0.05) in 17 of 22 pairs. This is ‘sampling error’ and is clearly a major source of inaccuracy when TLIs are derived from single small samples of heterogenous tumours such as cancers of the human breast.

Rate and time of DNA synthesis of human leukaemic blasts in bone marrow and peripheral blood.

We have evaluated DNA synthesis rate (S rate) and time (Ts) and tritiated thymidine labelling index (LI) of peripheral blood (PB) and/or bone marrow (BM) leukaemic blasts (Bl) in nineteen cases of acute leukaemia (twelve non-lymphoblastic, AnLL, and seven lymphoblastic, ALL), in one case of non-Hodgkin's leukaemic lymphoma and in a case of plasma cell leukaemia. The LI of PB-Bl was significantly lower than that of BM-Bl (range 0.1-6.2% and 1.9-19.5%, respectively; P less than 0.01). The S rate was higher for PB-Bl than for BM-Bl (range 3.5-11.3 and 2.5-9.5 mol X 10(-18)/min; P less than 0.02) and the Ts of PB-Bl was shorter than that of BM-Bl (range 7.6-22.1 and 10.8-34.7 hr, respectively; P less than 0.02). In eight cases where S rates of both BM-Bl and PB-Bl were available, a linear correlation (r = 0.82; P less than 0.01) was found between the two parameters. This suggests that the DNA synthetic rate is a property of the leukaemic cell line in individual patients and differs from case to case. It further indicates that the environmental influences on the DNA synthesis rate in BM or PB are always of the same order of magnitude. From the results of this study we speculate that the DNA synthesis rate of leukaemic blasts is slowed down in the BM by environmental factors such as cell density.

Development of cell and fiber lamination in the mouse superior colliculus.

The emergence of laminar organization in the superior colliculus was investigated in the mouse with several anatomical methods, including tritiated-thymidine autoradiography, Golgi impregnation, and general stains for cell bodies and for fibers. The sequence of neurogenesis, cell migration, and early morphological differentiation of neurons was shown to exhibit a discontinuity between the lower and upper divisions (i.e., between the deep and intermediate "gray" and "white" layers and the superficial "gray" and "white" layers). These events proceed in an inside-out order within the lower division, but the same events within the upper division commence in advance of the completion of this progression. Thus, peak generation times for layers of the lower division proceed from (embryonic day) E11 to E13 and for the upper division from E12 to E13. Cell migration, as monitored with tritiated-thymidine labelling, reflects closely the pattern of cytogenesis. This is most clearly evident on E15 when a population of E11-labelled cells is divided into superficial and deep layers (the strata superficiale and profundum--SS and SP) by the interposition of E13-labelled cells at an intermediate level (stratum intermedium--SI). A contingent of the latter cells continue their migration and join their predecessors within the SS on E17, a time point when cell migrations are largely complete. Paralleling this sequence of arrival of neurons and the formation of three primary layers, both the time course of accumulation of fiber fascicles and the early morphological differentiation of neurons in the interval from E13 to E17 tends to proceed from SP to SS and from SS to SI. Thus, the transverse fiber system and large multipolar neurons of SP develop in advance of the longitudinal fiber system and vertically oriented neurons of SS, which in turn develop precociously with respect to the longitudinal fibers and medium-sized multipolar neurons of SI. In contrast, later events of differentiation that underly a major radial growth and an architectonic sublamination of the primary strata proceed in a simpler inside-out sequence from E17 to (postnatal day) P6. The major morphogenetic events underlying the establishment of statification in the colliculus appear to involve the operation of relatively independent programs of assembly for the two basic subdivisions. It is probable that selective cell-cell interactions contribute to the delivery of concurrently generated neurons to different laminae as well as to the deployment of axons in a manner that respects laminar boundaries.

Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues.

A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. To examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. The number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. The observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.

Association of plasma intermediate density lipoproteins with atherogenic intimal proliferative activity in abdominal aortas of hyperlipidemic swine

In an earlier study of swine fed hyperlipidemic (HL) diets containing either butter or corn oil we found wide differences in atherogenic intimal proliferative responses among the swine, especially in the corn oil group, that could not be entirely accounted for by differences in serum cholesterol levels. We hypothesized that there might be differences in some lipoprotein constituents other than total cholesterol that might better account for the differences in intimal proliferative responses. In the current experiment, 11 swine were fed HL diets containing butter (n = 5) or corn oil (n = 6). At 90 days on HL diet, plasma was obtained for lipoprotein fractionation. At 120 days the swine were killed and the lesion areas and number of nuclear profiles in the intima or lesions in multiple cross-sections of the distal abdominal aorta were determined under light microscopy. Tritiated thymidine labeling indices of the intima or lesion cells were also determined. On comparing the butter and corn oil groups, only IDL-cholesterol and tritiated thymidine labeling indices of the cells in the lesions were significantly different. Based on the number of nuclear profiles per cross-section (Np/Cx) 7 were classified as high intimal proliferative responders (5 butter, 2 corn oil) and 4 as low responders (4 corn oil). The high-proliferative responders had plasma IDL levels 6-fold greater than those of the low responders with no overlaps. LDL, VLDL and HDL were not significantly different between the high- and low-proliferative responders. Among correlation coefficients between Np/Cx and plasma lipoprotein variables for all swine (n = 11), IDL cholesterol level was best correlated with Np/Cx. However, in the high responder group LDL values showed a highly significant correlation with the amount of intimal proliferation.

Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia.

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.

Differences between labeling index and DNA histograms in assessing S-phase cells from a homogeneous group of chronic phase CML patients.

The reliability of DNA histogram analysis in accurately estimating S-phase cells from human tumors was tested by comparing the results to those of simultaneously obtained tritiated thymidine labeling index (LI) studies. Patients with chronic myelocytic leukemia (CML) during chronic phase were selected for study because the Philadelphia chromosome (Ph) was the only cytogenetic abnormality in each case and, since it is a balanced translocation, the frequently encountered problem of aneuploidy in human neoplastic cells was avoided. Unfortunately, when 30 CML patients were studied simultaneously by DNA histogram analysis and LI studies, the correlation coefficient between the two results was only r = 0.611. A comparison of three different mathematical programs for DNA histogram analysis showed that none was completely satisfactory. We conclude that DNA histogram analysis does not provide the same data as autoradiographically processed labeling index studies even in patients with Ph-positive CML during the chronic phase when the situation is not complicated by additional aneuploidy.

Endothelial cell labeling indices in swine aortas in relation to intimal cell mass-derived atherosclerotic lesions

In the swine abdominal aorta, most atherosclerotic lesions arise from naturally occuring collections of intimal cells called intimal cell masses. The main objective of this study was to determine if the tritiated thymidine labeling index of endothelial cells lying over intimal cell masses was greater than that of endothelial cells not over intimal cell masses. A higher endothelial cell labeling index over intimal cell masses would indicate possibly a greater turnover in the area. Such a finding would suggest possible transient but repeated breaks in the endothelial cell barrier that might contribute to the initiation of the atherosclerotic lesion in intimal cell masses. In 2 groups of mash-fed swine, 2 and 11 months old, no differences were found in labeling indices of endothelial cells lying over or not over intimal cell mases. It appears that the initiation of atherosclerosis in swine abdominal aorta is not dependent upon pre-existing altered endothelial cell kinetics as measured by tritiated thymidine labeling indices. The labeling index of cells within the intimal cell mass was significantly higher than in the media; this also is obviously not dependent upon an increased labeling index of the covering endothelial cells. Another group of swine were killed at 11 months of age after receiving a hyperlipidemic diet for 270 days. The labeling index of endothelial cells over atherosclerotic lesions was significantly higher than the labeling index of endothelial cells not over lesions in these swine, and also significantly higher than that of endothelial cells over intimal cell masses in the 11-month old mash-fed group. It is concluded that the increased endothelial cell labeling index over atherosclerotic lesions may be secondary to the lesion itself, and not due to the direct effect of the hyperlipidemic blood.