Tag Archives: follicle (Part 4)

Vascular Remodeling and Angiogenesis: RESULTS(3)

Kinetic Studies of Intramuscular Ovarian Transplantation

The subcutaneous grafts showed extensive damage during the first days after transplantation. Therefore, the intramuscular grafts were assessed at different time periods, from 1 to 31 days posttransplantation (Fig. 3). Half ovaries were transplanted into the gluteus superficialis at the hind limb. Of the 31 grafts, 26 were detected (Table 1). One of the ovaries retrieved 24 h after transplantation was found to be disconnected from the muscle (Fig. 3, A-C). In that ovary, both the follicles and the vasculature were extensively damaged. Similar to the subcutaneous transplants, at this early time point after transplantation, damage was also detected in ovaries that were in close contact with the muscular tissue. buy glucovance

Vascular Remodeling and Angiogenesis: RESULTS(2)

RESULTS(2)Intramuscular Ovary Transplantation: Optimization of Graft Size

Because the subcutaneous region is heterogeneous and relatively poor in its blood vessel support, we chose the muscle, which is more homogeneous and rich with vasculature, as a transplantation site. While the graft (in muscle as well as in the subcutaneous transplantations) should be large enough to contain the maximum pool of oocytes, it should also be small enough to minimize ischemia-reper-fusion injury. In order to optimize the graft size in the intramuscular transplantation, we studied various sizes of ovarian grafts, ranging from intact ovaries (6 mm3) to 1/8 ovary (0.75 mm3; Fig. 2).

Vascular Remodeling and Angiogenesis: RESULTS(1)

Subcutaneous Ovarian Transplantation

Ovaries of 15-day-old Wistar rats contain primordial, primary, and small antral follicles (Fig. 1A). The GSL-1 staining represents staining of endothelial cells (e.g., Fig. 1B) and aSMA staining represents SMC and pericytes (e.g., Fig. 1C).

Subcutaneous half ovarian grafts were examined at various time points during the first days after transplantation (Fig. 1, D-L). Out of the 13 grafts, only 6 provided identifiable follicles within the ovarian sections (Table 1). A gradual decrease in the overall preservation (follicle state, vascular integrity, and necrosis) of the ovarian grafts was observed. micronase dosage

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(7)

METHODS(7)

We have applied a relative scale in order to estimate the condition of the ovary on the basis of follicle growth and viability. The seven follicle types were scored as follows: the viable ones received a positive grade from 1 to 7; the smallest one received a score of 1 and the largest one a score of 7. The atretic follicles were negatively scored, the smallest one received a score of —7 and the largest one received a score of —1. The rationale for this scoring system is based on the probability that small follicles being atretic is normally low, while as the follicles grow, their probability of being atretic is increased. Therefore, we assume that excessive atresia of small follicles in the transplants is the result of ovarian stress due to insufficient blood supply. Thus, atretic primordial follicles received the most negative score (—7). Viable antral follicles received the highest positive score (+7). Each follicle type was graded as follows: follicle grade X (total number of follicles from the specified type/total area of the examined ovary [mm2]). The total score of an ovary was obtained by summing up the scores of each follicle type.

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(6)

Follicle Examination

The eosin-hematoxylin-stained slides were used for follicle counts. The follicles were counted in all the detected grafts (Table 1). Both viable and atretic follicles were counted. Follicles were considered atretic if more than 1% pyknotic nuclei were found in the granulosa cells or if the oocyte began to degenerate. In order to avoid double counting of the same follicle and to assure inclusion of the largest cross-sections of follicles, the counts were performed in the section in which the nucleolus was within the germinal vesicle (oocyte nucleus). buy prandin

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(5)

METHODS(5)

Two out of each three slides of the subcutaneous transplants and every third slide in the intramuscular transplants were stained by eosin-hema-toxylin, while the other slides were stained for endothelial cells, for smooth muscle cells, and for the biotinylated MRI contrast material. The paraffin-embedded unstained sections were deparaffinized with xylene for 5 min, followed by sequential ethanol hydration and double distilled water. cheap prozac

Endogenous peroxidase was inactivated with 3% H2O2 in PBS for 5 min at room temperature. Sections were then washed with PBS for 5 min and were blocked by overnight incubation in 1% BSA in PBS at 4°C.

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(4)

The concentration maps were used for derivation of vascular permeability (apparent permeability surface area product, APSMRI), namely the rate of contrast accumulation, derived by linear regression of the first 10 min of postcontrast agent administration. Only pixels with significant regression (r > 0.9) were considered. Data are presented using a color scale for APSMRI values between 0 and 0.8 ^M/min, overlaid on a gray-scale anatomical image. Mean changes in APSMRI were calculated from selected regions of interest, mainly the graft and the adjacent muscle.

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(3)

METHODS(3)

BSA-based macromolecular contrast material, biotin2-BSA-gadolini-um-DTPA23 (biotin-BSA-GdDTPA; about 82 kDa) was prepared as reported previously and bolus injected through a tail vein catheter (12.4 mg/mouse in 0.2 ml).

A series of precontrast spin echo images, with repetition time (TR) values of 1000, 500, 200, and 100 msec, were acquired to determine the precontrast R1. For dynamic postcontrast imaging, T1 weighted spin echo images were obtained 4-32 min after the contrast agent was administered (TR 200 msec, echo time 10.6 msec, two averages, spectral width 50 000 Hz, field of view 35 mm, slice thickness 1 mm, matrix 256 X 256, in plane resolution 137 |xm, acquisition time 105 sec).

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(2)

In all transplantations, one ovary piece was transplanted into each mouse. In the subcutaneous transplantation, half ovaries were transplanted into 13 mice (Table 1). The ovaries were retrieved between Days 1 and 24 after transplantation. In the intramuscular ovarian transplantation, half ovaries were transplanted into 31 mice (Table 1). The grafts were retrieved between Days 1 and 31 after transplantation. In addition, different sizes of intramuscular ovarian grafts, obtained by cutting the ovary by fine tweezers under a binocular microscope, were studied by MRI (see below) and retrieved after 5-35 days. The grafts ranged from intact (6 mm3) to 1/8 ovary (0.75 mm3; Table 1). cheap wellbutrin

Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(1)

METHODS(1)Ovary Retrieval and Transplantation

All animal experiments were approved by the Institutional Animal Care and Use Committee. Immature 15-day-old Wistar rats were killed using CO2 and subsequent cervical dislocation, and ovaries were collected. The ovaries were cleaned from fat in L-15 Leibovitz medium (GibcoBRL, Invitrogen Corporation, Paisley, Scotland, UK) supplemented with 0.1% BSA and antibiotics (penicillin/streptomycin; 100 IU/ml) at room temperature. Several cleaned ovaries were fixed for morphological evaluation and served as controls. Ovaries were transplanted into CD-1 female nude mice, 6-10 wk old, within 20-30 min after collection. Briefly, mice were anesthetized by intraperitoneal administration of 75 mg/kg Ketaset (ketamine; Fort Dodge Laboratories, Fort Dodge, IA) and 3 mg/kg XYL-M (xylazine; VMD, Arendonk, Belgium) followed by a subcutaneous addition of approximately half of the initial dose in order to prolong the duration of anesthesia. Ovarian fragments were transplanted either subcutaneously at the hind limb above the gluteus superficialis muscle or in the muscle itself.

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