Lymphatic Density Research Articles

D2-40 positive lymphatic endothelial cell density is much higher in and along the mammary glandular tissues than in subcutaneous tissue: Case report of a 20-year-old donated female cadaver

Sappey's demonstration of the breast lymphatic vessels, showing the presence of a subareolar lymphatic plexus and few thick vessels originating from the breast itself, is famous. However, with the exception of such studies performed by injection methods, to the best of our knowledge there has been no demonstration of the entire distribution of the breast lymphatic vessels. D2-40 immunohistochemistry of semiserial sections (at 0.1-mm intervals) taken from the healthy breast of a 20-year-old female cadaver revealed that the lymphatic endothelial density was much higher in and along the mammary glandular tissues than in the overlying subcutaneous and deep fascial tissues. Thus, the extent of the subareolar lymphatic plexus is likely to have been overestimated by the injection method. Instead, networks of lymphatic vessels around the mammary glands were evident in the physiological state.

Microvessel and lymphatic density in tubular carcinoma of the breast: Comparative study with invasive low-grade ductal carcinoma

Tubular carcinoma (TC) of the breast is an uncommon variant of ductal carcinoma, which has an extremely low metastatic potential and an excellent prognosis. Studies concerning the vascular and lymph vessel status in TC are not numerous and hampered by a lack of specific markers. We immunohistochemically analysed the expression of CD31 and D2-40 antibodies in 15 TC measuring less than 2cm in diameter. We compared the results with those observed in 30 low-grade ductal breast carcinomas (LGDC) of no specific type with similar dimensions. Neither microvascular density (MVD) nor lymphatic vessel density (LVD) was statistically different in the groups: MVD was 26.5+/-28.2 and 32.1+/-17.9 vessels/mm(2) in TC and LGDC, respectively (P=0.08). LVD, low in both tumour types, was 0.5+/-0.3 and 0.6+/-0.4 vessels/mm(2), respectively (P>0.1). These data suggest that the favourable prognosis and the low rate of lymph node metastases in TC are not explained by lymphangiogenetic and angiogenetic potential and are supported by recent analyses in the literature.

Dysadherin Expression in Head and Neck Squamous Cell Carcinoma

Dysadherin is a recently characterized cancer-associated cell membrane glycoprotein that has a crucial role to cell-cell adhesiveness. The aim of this study was to examine dysadherin expression in head and neck squamous cell carcinoma (HNSCC). A total of 108 tissue specimens of patients with HNSCC were examined using immunostaining for dysadherin, E-cadherin, and the specific lymphatic endothelium marker D2-40. We quantified dysadherin and E-cadherin expression, assessed intratumoral (ILD) and peritumoral lymphatic density (PLD), and examined the possible associations of all the above parameters with clinicopathologic features and outcome. Finally, we used double staining with dysadherin and D2-40 to examine the expression pattern of dysadherin simultaneously with the lymphovasculature environment of HNSCC. High dysadherin expression was correlated with higher clinical stage (chi2, P = 0.01), with the presence of lymph node metastasis at the time of diagnosis (chi2, P = 0.02), and with increased ILD (chi2, P = 0.001). We observed an impressive reverse association between increased dysadherin expression and decreased E-cadherin expression (chi2, P < 0.001). Surprisingly, dysadherin-positive cancer cells usually gathered around areas of high intratumoral lymphatic vessel concentration, surrounding and invading small intratumoral lymphatics. Higher clinical stage and increased dysadherin expression were found to be the only significant independent prognostic factors for overall survival (hazard ratio, 3.94; 95% confidence interval, 1.09-14.27 for clinical stage; hazard ratio, 3.92; 95% confidence interval, 1.46-10.51 for dysadherin). The loss of intercellular adhesiveness and increased dysadherin expression seems to be related to lymphangiogenesis in HNSCC, but this should be confirmed by additional studies. Dysadherin expression might be a promising prognostic marker for separation of patients at higher risk.

First year changes of myocardial lymphatic endothelial markers in heart transplant recipients☆

The lymphatic system plays an important role in interstitial fluid balance, lipid metabolism, and immune response. The recent introduction of specific lymphatic endothelial cell markers has made the investigation of lymphangiogenesis under various conditions and from small tissue samples feasible. It was the purpose of the study to investigate the changes of myocardial lymphatic endothelial markers during the first 12 months after heart transplantation and to analyze if a correlation between lymphatic markers and rejection can be found. Right ventricular endomyocardial biopsies taken for routine rejection monitoring from 26 heart transplant recipients were investigated. Selected time points were 0.5, 1, 1.5, 6, and 12 months after human heart transplantation (HTX). Immunohistostaining was performed for VEGFR-3, the receptor for lymphangiogenic vascular endothelial growth factors C and D, for LYVE-1, a novel hyaluronan receptor, restricted to lymphatic vessels, and PROX-1, a homeobox gene product, which plays a key role in lymph vessel development and differentiation. Density of VEGFR-3 positive lymphatics did not change during the first 12 months after transplantation. However, in comparison to the 0.5-month biopsy, density of LYVE-1 and PROX-1 positive lymphatics was significantly decreased at 1 month after transplantation (p<0.03) and at the subsequent time points (p<0.01). Patients with only moderate rejection during the first 12 months (ISHLT<IIIa) had a significantly higher density of VEGFR-3 at 0.5 month in comparison to patients with at least one episode of clinically relevant rejection (ISHLT IIIa or worse, p<0.03). Myocardial lymphatics show a significant change of endothelial phenotype after transplantation, as demonstrated by significant quantitative changes of lymphatic endothelial marker density. Patients with at least one rejection of ISHLT IIIa or higher had a significantly lower density of VEGFR-3 at 0.5 month after transplantation. The results of this study warrant further investigation on the impact of transplantation on the lymphatic endothelium. The cause-effect relation between rejection and lymphatic endothelium remains to be investigated.

Lymphatic Density and Metastatic Spread in Human Malignant Melanoma

Lymphatic Density and Metastatic Spread in Human Malignant Melanoma

Comparison of the diagnostic accuracy of lymphatic endothelium markers: Bayesian approach

Tumor lymphatic density is evaluated by means of specific lymphatic endothelium markers, and is a potential predictor of clinically meaningful outcomes. There are many claims on the postulated superiority of some of these markers to identify lymphatics, always in the absence of quantitative data. We therefore compared the diagnostic accuracy of the antibody against podoplanin and the commercially available D2-40, employing Bayesian statistics to account for the absence of a gold standard. We used the pan-endothelial marker CD34 to identify 23 542 distinct blood and lymphatic vessels in sections from 30 formalin-fixed, paraffin-embedded archival tissue blocks of head and neck squamous cell carcinoma specimens. We stained two adjacent sections with podoplanin and D2-40 and identified the continuum of each stained vessel in the sections with a comprehensive method. Overall, 1864 vessels were stained with both markers, 119 only with podoplanin and 391 only with D2-40. Significantly more vessels with intraluminal red blood cells were stained with D2-40 compared to podoplanin (McNemar’s P<0.0001). Both antibodies had extremely high specificity (99.7% (95% credible interval (CrI): 99.5–99.9%) and 98.8% (95% CrI: 98.3–99.5%) for podoplanin and D2-40, respectively) and very high sensitivity (92.6% (95% CrI: 86.1–97.9%) and 97.3% (95% CrI: 94.9–99.2%) for podoplanin and D2-40, respectively). Inferences were qualitatively similar when we took into account in the analyses the possibility that the two tests (antibodies) may be correlated. We calculated that 96.3% (95% CrI: 94.2–98.6%) of the vessels stained with podoplanin and 88.9% (95% CrI: 83.9–95.7%) of the vessels stained with D2-40 were truly lymphatics. These numbers were in agreement with the observed number of stained vessels without intraluminal red blood cells. Our results suggest that both antibodies are excellent lymphatic endothelium markers and that there may be little reason to prefer either of them in most settings.

Targeting of pericytes diminishes neovascularization and lymphangiogenesis in prostate cancer

The walls of capillaries in prostate cancer are composed of endothelial cells, and pericytes. NG2 is a transmembrane proteoglycan on nascent pericytes with a functional role in neovascularization. The anti-angiogenic effect of hydron pellets containing NG2 neutralizing antibody was quantified in intracorneal PC-3 and LNCaP xenografts. TRAMP and TRAMP-C1 tumors grafted in NG2 knockout mice represented intrinsic pericyte targeting. TRAMP and TRAMP-C1 grafts were analyzed with confocal microscope for microvascular density (MVD) and lymphatic vascular density (LVD). NG2 neutralizing antibody decreased corneal neovascularization in PC3 (P<0.0001), and LNCaP (P=0.0079) xenografts. Mean MVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 71% (P=0.0006) and 63% (P=0.0011) lower than wild type controls, respectively. Mean LVD in TRAMP and TRAMP-C1 tumors in NG2 knockout mice were 73% (P=0.0003) and 84% (P<0.0001) lower than wild type controls, respectively. Targeting of pericyte-NG2 decreases neovascularization and lymphangiogenesis in prostate cancer significantly.

Overexpression of VEGF-C Causes Transient Lymphatic Hyperplasia but Not Increased Lymphangiogenesis in Regenerating Skin

Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis and holds potential for lymphangiogenic therapy in diseases lacking adequate lymphatic drainage. However, the ability of VEGF-C to enhance sustainable, functional lymphatic growth in adult tissues remains unclear. To address this, we evaluated VEGF-C overexpression in adult lymphangiogenesis in regenerating skin. We used a model of mouse tail skin regeneration incorporating a suspension of either VEGF-C overexpressing tumor cells, which provide a continuous supplement of excess VEGF-C to the natural regenerating environment for more than 25 days, or otherwise identical control-transfected tumor cells. We found that excess VEGF-C did not enhance the rate of lymphatic endothelial cell (LEC) migration, the density of lymphatic vessels, or the rate of functionality -- even though lymphatic hyperplasia was present early on. Furthermore, the hyperplasia disappeared when VEGF-C levels diminished, which occurred after 25 days, rendering the lymphatics indistinguishable from those in control groups. In vitro, we showed that whereas cell-derived VEGF-C could induce chemoattraction of LECs across a membrane (which involves amoeboid-like transmigration), it did not increase LEC chemoinvasion within a 3-dimensional fibrin matrix (which requires proteolytic migration). These results suggest that whereas excess VEGF-C may enhance early LEC proliferation and cause lymphatic vessel hyperplasia, it does not augment the physiological rate of migration or functionality, and by itself cannot sustain any lasting effects on lymphatic size, density, or organization in regenerating adult skin.

Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma

Lymph node metastasis is a frequent reason for adverse clinical outcome in many epithelial neoplasms, including head and neck squamous cell carcinoma. The mechanisms underlying the capability of epithelial neoplasms to metastasize via lymphatic vessels have not yet been fully elucidated. There is great debate about whether cancer cells can metastasize by expansion and invasion of pre-existing peritumoral lymphatics or by the formation and invasion of new lymphatics within tumours (lymphangiogenesis). In order to investigate this issue, we examined 81 tissue specimens from patients with head and neck squamous cell carcinoma, using immunostaining for the specific lymphatic endothelium marker podoplanin, and assessed intratumoral and peritumoral lymphatic density. We also quantified lymphatic invasion and examined the possible associations of all the above parameters with clinicopathological features and outcome. Finally, we used double staining with podoplanin and the cell proliferation marker Ki-67 in order to evaluate lymphangiogenesis. High intratumoral and peritumoral lymphatic density were both significantly associated with the presence of lymph node metastasis at the time of diagnosis (chi2 test, p < 0.001 and p = 0.007, respectively) and there was a significant correlation between high intratumoral lymphatic density and lymphatic invasion. Patients with higher intratumoral lymphatic density exhibited shorter overall survival (log rank p < 0.001) and this correlation remained significant after multivariate analysis (Cox p = 0.04), indicating that intratumoral lymphatic density is an independent prognostic factor for mortality. Peritumoral lymphatic density had no influence on outcome. Double staining revealed the existence of proliferating intratumoral lymphatics, in which tumour emboli were occasionally observed. These results indicate that lymphangiogenesis indeed occurs in head and neck squamous cell carcinoma; that newly formed vessels are targets of invasion by cancer cells; and that intratumoral lymphatic density might be used as a criterion to separate patients at higher risk of an adverse clinical outcome.

285. Endometrial lymphatics are reduced in the functionalis compared to basalis and myometrium

Information on uterine lymphatics is limited. The aim of this study was to characterise uterine lymphatic vessels and the corresponding growth factors in the endometrium and myometrium across the normal human menstrual cycle. Uterine tissues were collected from patients undergoing hysterectomy. Lymphatic and microvascular density (MVD/mm2) was determined on serial sections of full thickness uterus (n = 45) with antibodies to D2-40, CD31, CD34 and FVIII. Lymphangiogenic growth factors VEGF-C and VEGF-D immunolocalisation was also determined on serial sections. VEGF-C, VEGF-D and lymphatic endothelial cell markers VEGF-R3 and D2-40 protein expression was determined on protein extracted from myometrium and endometrium and separated by SDS-PAGE from proliferative (n = 5) and secretory (n = 5) hysterectomy specimens. The lymphatic vessels were closely associated with smooth muscle cells of spiral arterioles and VEGF-C and VEGF-D were primarily localised in the endometrial glands, luminal epithelium and myometrial smooth muscle bundles. The lymphatic MVD was significantly reduced in the functionalis (15.1 ± 2.3mm2) compared to basalis (80 ± 11.4mm2) and myometrium (63 ± 9.2mm2). Overall, lymphatics constituted 12% of all vessels in the functionalis, 60 % in the basalis and 30% of the myometrium. D2-40 positive uterine lymphatics showed considerable heterogeneity, with 88% co-localisation with the blood vessel marker CD31, but only 46% expressing CD34 and 31% with FVIII. Protein expression of VEGF-C, VEGF-D, VEGF-R3 and D2-40 were significantly reduced during the proliferative phase compared to the secretory phase and were also significantly reduced in the endometrium compared to the myometrium across the cycle (P ≤ 0.05). Endometrial functionalis lymphatics are reduced in conjunction with a reduction in lymphangiogenic growth factors compared with the myometrium.

Lymphatic vessel density is significantly increased in melanoma

Melanoma is well known for its ability to involve regional lymph nodes in the early stage. However, the presence of lymphangiogenesis in melanoma is still controversial due to lack of lymphatic-specific markers. The purpose of this study was to determine the intra- and peritumoral lymphatic vessel density (LVD) using a novel lymphatic vessel-specific marker D2-40 and compare it to general vessel density (GVD) as determined by CD31 immunostaining in a series of melanocytic lesions. The intra- and peritumoral GVD and LVD were examined by immunohistochemistry using D2-40 and CD31 antibodies in a series of melanocytic lesions. We found significantly higher intratumoral LVD in melanomas as compared to either common acquired or dysplastic nevi (p < 0.01). Although peritumoral LVD in melanoma and malignant melanoma in situ was higher compared to nevi, the difference did not reach statistical significance (p = 0.059). There was no significant difference in GVD among the various groups of melanocytic lesions. Our results show that intratumoral LVD is significantly increased in melanomas compared to benign nevi. The higher intratumoral lymphatic density in invasive melanomas suggests that melanoma cells might promote lymphangiogenesis. In addition, assessment of intratumoral LVD may be potentially useful in the differential diagnosis of melanocytic lesions.

Angiogenesis and lymphangiogenesis in parathyroid proliferative lesions.

Angiogenesis and lymphangiogenesis are involved in tumoral growth and metastatic spread. There is little information on angiogenesis and no available data on lymphangiogenesis in parathyroid glands (PTG). Using immunohistochemistry for CD34, LYVE-1 (specific markers for vascular and lymphatic endothelium, respectively), vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, this study analyzes microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic growth factors in 13 normal PTG, 77 parathyroid adenomas (PTA), and 17 primary parathyroid hyperplasia (PPH). MVD was higher in PPH and PTA, compared with PTG (P < 0.001). There was no difference in VEGF-A expression among groups. In contrast, FGF-2 expression was higher in PPH, compared with PTA and PTG (P < 0.0001). FGF-2 scores and MVD were significantly correlated (r = 0.43). LVD did not differ among groups, and VEGF-C expression was unrelated to LVD. There was no relationship between MVD and tumor behavior (adenoma size, PTH, or calcium). In conclusion, this study shows increased angiogenesis in parathyroid proliferative lesions compared with normal glands and suggests that FGF-2 is proangiogenic in parathyroid tissue. In PTA, tumor behavior is not related to angiogenic phenotype. This is the first demonstration of lymphatic vessels in PTG, but the lack of correlation with VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic factor.

Morphology and density of initial lymphatics in human myocardium determined by immunohistochemistry.

Myocardial edema is a common finding in congestive heart failure, transplant rejection, and cardiomyopathy. Although pathological alterations in the lymphatic morphology are part of the underlying disease, knowledge on these changes is limited. However, lymphatic morphology may be investigated by immunohistochemical staining for fms-like tyrosine 4 kinase (flt-4), which is specific for lymphatic endothelium in adult tissue. We used myocardial tissue of ventricular out flow tract taken from five human semilunar valves harvested as allografts but unsuitable for implantation for analysis, performing immunohistochemical staining for flt-4 with a commercially available antibody. Lymphatic morphometry was completed according Gundersen method. Immunohistochemical staining for flt-4 resulted in successful labeling of lymph capillaries in adult human myocardium. Lymph capillary density was calculated as 50.7 +/- 12.5 per mm2 and average diameter was 3.7 +/- 0.7 microm. Conclusions. Lymph capillary morphology in human myocardium may be successfully determined by immunohistochemical staining for flt-4. Tissue samples as small as myocardial biopsies may be used for analysis. Using this method, morphological changes in myocardial lymphatics may be investigated in various cardiovascular diseases.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Anatomic distribution of intraprostatic lymphatics: implications for the lymphatic spread of prostate cancer-a preliminary study.

Although prostate cancer metastasizes primarily into the lymphatic system, the anatomic distribution of intraprostatic lymphatics has not been elucidated. The aim of this study was to clarify the distribution of lymphatics in the human prostate by immunohistochemical techniques, using an anti-desmoplakin antibody. Whole-mount cryostatic sections were serially cut from the noncarcinomatous prostates of adult men with bladder carcinoma who underwent cystoprostatectomy. Each section was stained either with a monoclonal antibody specific for desmoplakin or with hematoxylin and eosin. Lymphatics were detected in all components of the prostate. In the glandular prostate, lymphatics were distributed evenly in the peripheral, transitional, and central zones. The lymphatic density was significantly high in the midbase region surrounding ejaculatory ducts. These results may indicate the importance of the midbase region as a route of lymphatic spread of prostate cancer.

Increased Dermal Lymphatic Density in the Human Leg Compared with the Forearm

During orthostasis, capillary filtration pressure is higher in the leg than in the arm due to the effect of gravity. We investigated the possibility that the lymphatic network in leg skin might be adapted to cope with a greater fluid load. The dermal lymphatics of the forearm and lower leg were studied in white-skinned and brown-skinned men using fluorescence video microscopy. From video print lymphangiograms the following were determined: lymphatic length density at a series of radii from the centre of the lymphangiogram (LDr); maximum lymphatic density (LDmax); total length of lymphatic vessel (LL); maximum spread of lymphatic vessel; number and size of lymphatic rings (continuous circuits of vessel); and vessel diameter. There were no differences between the two racial types, but clear differences between the arm and leg. In the leg, mean (± SD) peak LDr(25.13 ± 5.65 cm−1), LDmax(32.95 ± 6.89 cm−1), LL (40.17 ± 27.42 cm), and spread (1.39 ± 0.08 cm) were all significantly higher than in the arm (18.03 ± 5.48 cm−1, 23.91 ± 7.21 cm−1, 11.76 ± 5.47 cm, and 1.00 ± 0.05 cm respectively,P≤ 0.001). In the region of maximum lymphatic density there were 2.4 times more rings in the leg than in the arm, ring size being similar. Vessel image diameters were 89.6 ± 26.3 μm (leg) and 94.8 ± 10.9 μm (arm). Leg skin thus has a denser and more extensive lymphatic network than arm skin. This supports the hypothesis that lymph transport capacity in the leg is enhanced to compensate for a greater gravitational fluid loading.

Arm and leg skin lymphatic density studied in different racial types using quantitative fluorescence microlymphography

Arm and leg skin lymphatic density studied in different racial types using quantitative fluorescence microlymphography

Quantification of the Initial Lymphatic Network in Normal Human Forearm Skin Using Fluorescence Microlymphography and Stereological Methods

Fluorescence microlymphography enables the visualization of the initial lymphatic network in human dermisin vivo.In order to determine which objective, measurable parameters are likely to be of value in future studies, microlymphangiograms were obtained from normal forearm skin of eight men and nine women of a range of ages and analyzed stereologically to calculate: (1) lymphatic length density at a specified radial distance,r, from the site of injection of the fluorescent dye (LDr, cm−1); (2) maximum lymphatic density (LDmax, cm−1); (3) total length of filled lymphatic (LL, cm); (4) maximum radial spread of vessels (mm). There was no significant difference between the right and left arms for peak LDr, LDmax, LL, or spread. Mean values (±SD) for the right and left arms combined (n= 24) were 13.34 ± 7.61 cm−1; 19.31 ± 8.96 cm−1; 12.04 ± 10.04 cm; and 7.58 ± 3.30 mm, respectively. Unexpectedly, three of the four parameters were significantly lower in women compared to men. For women LDmaxwas 30% lower than for men (P= 0.04), LL was 59% lower (P= 0.032), and spread was 39% lower (P= 0.007). Peak LDrdid not differ significantly. Stereological analysis of fluorescent microlymphangiograms enables quantification of the dermal initial lymphatic network and hence the objective study of the influence of gender, age, and disease (including treatment interventions).

Morphological indications for considerable diffuse reabsorption of cerebrospinal fluid in spinal meninges particularly in the areas of meningeal funnels. An electronmicroscopical study including tracing experiments in rats.

Transmission and scanning electron microscopical observations in the rat indicate a considerable capacity of the spinal meninges to reabsorb cerebrospinal fluid. The density of blood vessels and lymphatics in the duramater is extremely high, particularly in the areas of meningeal funnels and spinal nerve root sleeves. Arterioles with closely related unmyelinated nerve fibres, many fenestrated capillaries and venules predetermine these areas as sites where absorption processes could take place. At certain sites of the meningeal angle region, the arachnoid membrane, mostly multilayered, is reduced to only three or four layers. Intercellular discontinuities and cytoplasmic fenestrations occurring in the arachnoid lining cell layer result in direct communications between the subarachnoid space and cisterns of the arachnoid "reticular layer". These cisterns are partly fluid-filled, partly occupied by a net of collagen fibre bundles. Some cisterns harbour macrophages that often project filiform processes through the lining cell layer into the subarachnoid space, contacting cerebrospinal fluid. Desmosomes and gap junctions are present in all layers of the arachnoid. However, tight junctions and the continuous electrondense intercellular gap, known to occur normally within the "arachnoid barrier layer", were not seen in many sites of the meningeal angle region. Numerous arachnoid cells display a high degree of vesiculation. Cationized ferritin, introduced in vivo into the rat subarachnoid space, passes inter- and intracellularly from the cerebrospinal fluid compartment through the arachnoid membrane, reaching dural blood vessels and lymphatics. Tracer could be visualized both in the cytoplasm of the endothelium and on the luminal surface of the cells. Tracer also passed through pial cell layers into pial vessels, through leptomeningeal sheaths into vessels crossing the subarachnoid space, into the connective tissue compartment and into vessels of spinal dorsal root ganglia. In the angle region, a particularly large number of macrophages can be found on the surface of leptomeninges, within the arachnoid reticular layers, and in close relation to dural and epidural capillaries, venules and lymphatics. Their possible role in the process of cerebrospinal fluid reabsorption is discussed.

Initial lymphatics are present in the loose areolar connective tissue of the golden hamster's cheek pouch

The immunologically privileged status of the hamster cheek pouch has been attributed to both the loose, areolar connective tissue that lines the pouch and to the lack of lymphatics in it. Although early reports described an absence of demonstrable lymphatics by classical histologic and lymphangiographic methods, issue has been taken recently as to whether the cheek pouch is alymphatic. We became interested in this issue accidentally during a study of microembolic microvascular injury. Our results, which were obtained by the coordinated use of intravital, light, and transmission electron microscopy, showed that a small population of initial lymphatics are present in the loose, areolar connective tissue. The areal density of initial lymphatics in 12 cheek pouches averaged 1.5 lymphatics/cm 2 of pouch connective tissue. We conclude that the hamster cheek pouch is drained by a small population of initial lymphatics, the general paucity of which probably contributes to the delayed egress of antigens to regional lymph nodes.