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A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Children's Hospital at Montefiore

The purpose of this study was to investigate the effect of oral administration of manganese acetate on the kidneys and urinary bladder of Sprague-Dawley (SD) rats. Male and female SD rats (150 to 175 g), 6 weeks old, were administered varying doses of manganese acetate for 63 days by oral gavage. At the end of 63 days, 50% of the animals were sacrificed and kidney tissue was isolated and fixed for histopathological studies (study A). The remaining 50% were cross-mated and dosing ceased. Animals were sacrificed after 2 weeks (study B). Male treated animals were noted to have viscous, gritty urine in the urinary bladder, and the high-dose groups had urinary bladder stones (uroliths). Histopathologically, the most striking lesions were observed in the kidneys and prostate glands of male animals. Mild-to-moderate tubulointerstitial nephritis with tubular proteineous and glomerulosclerosis was observed in animals of all treatment groups. Urolithiasis in the urinary bladder was confirmed in 33% to 66% of treated animals. Female animals did not show a significant difference above controls in renal tissues. Results of this study suggest that male rats are more sensitive to the effects of high levels of manganese given orally than female rats and that the genitourinary structures represent target organs of toxicity.

Effects of oral administration of manganese on the kidneys and urinary bladder of Sprague-Dawley rats.

Manganese has wide industrial applications and exposure to manganese can result in serious health conditions. The purpose of this study was to determine the reproductive effect of oral manganese exposure in male mice. Manganese acetate was tested at three dose levels (7.5, 15.0, and 30.0 mg/kg/day) for 43 days. The control group (0 mg/kg/day) received distilled water. Control negative group did not receive anything. Reproductive organ weights were recorded. Histopathology was performed on right testis, epididymis, seminal vesicle, and the accessory glands. Cauda epididymal, testicular sperm counts, and sperm motility was evaluated on the organ from the left side. The results of this study suggest that exposure to manganese caused a statistically significant (P<0.001) decrease in sperm motility and sperm counts at 15.0 and 30.0 mg/kg/day. There were no alterations in the fertility or pathology of the testicular tissue in the manganese-treated mice when compared with the controls.

Assessment of male reproductive system in the CD-1 mice following oral manganese exposure.

Our goal was to determine if breastfeeding provides any protection against urinary tract infection (UTI) and if vitamin D supplementation imposes any additional risks for UTI in infants < 3 months of age. In this study, 40% of the children who had urine cultures were breastfed, and 18.7% of the children were exclusively breastfed. Twenty percent of all of the urine cultures tested positive, and this number was greater in females (22.5%) than in males (18.1%, P < .05). There was no significant difference between the rates of positive urine cultures in exclusively breastfed (22% vs 21%, nonsignificant [NS]) formula-fed infants. The relative risk of UTI with breastfeeding versus formula feeding was 1.03 (0.58-1.82), and any breastfeeding versus no breastfeeding was 0.92 (0.58-1.45). Vitamin D supplementation increased the UTI risk, with a relative risk of 1.76 (1.07-2.91, P < .05). However, only formula-fed infants showed an increased risk of UTI after vitamin D supplementation.

Breastfeeding does not protect against urinary tract infection in the first 3 months of life, but vitamin D supplementation increases the risk by 76%.

Dual Energy X-ray Absorptiometry (DXA) is effective in measuring bone mineral density (BMD) in mice for early detection of osteoporosis. However, scanners designed for use with small animals (i.e. PIXImus) are very expensive. Used human DXA machines are cheaper to obtain, but analysis of scans from these instruments is operator-dependent. Obtaining reliable data depends on having a single operator analyze the scans in a blinded fashion. Scan quality is improved by excising the bone prior to scanning, which does not allow serial measurements. This study describes a novel method of analyzing lumbar spine BMD in mice using whole body DXA. This non-invasive technique has a high degree of precision and reproducibility, with good correlation between multiple observers. Inter-observer variability (0.063 +/- 0.00317 g/cm(2) [mean +/- SD], 5.05 [% coefficient of variation (CV)], repeat scan variability (0.063 +/- 0.00364 g/cm(2) [mean +/- SD], 5.94 [%CV]) were very low compared to variability between different animals (0.063 +/- 0.00588 g/cm(2) [mean +/- SD], 9.64 [%CV]) and variability seen in same animal over time (0.011 +/- 0.00885 g/cm(2) [mean +/- SD], 80.68 [%CV]). The measurement error is thus smaller than the biological variation. Accuracy was determined by comparing average peak BMD from two scans per mouse in-vivo (0.066 g/cm(2)) versus excised spine (0.065 g/cm(2)). Furthermore, correlation between bone ash weights and whole body lumbar spine BMD measurements (p < 0.0001) was highly significant. This technique thus shows a high degree of precision and accuracy, even with multiple observers, for measuring BMD in mice using a DXA machine designed for clinical use.

A new technique for precisely and accurately measuring lumbar spine bone mineral density in mice using clinical dual energy X-ray absorptiometry (DXA).

This study was conducted to determine if vitamin D supplementation is required to prevent rickets in breast-fed infants. Breast-feeding rates are increasing, and there are concerns about whether the vitamin D content of breast milk is sufficient. There are a few treatment trials of vitamin D supplementation in breast-fed infants; these were conducted in northern climates. The authors therefore performed a prospective clinical trial comparing vitamin D supplementation with placebo as control in southern Louisiana. Blood samples and questionnaires were collected at birth, 2, 4, and 6 months of age. There were no cases of rickets observed, and no differences in alkaline phosphatase levels between groups. Thus, there was no evidence that vitamin D supplementation reduced rickets risk in the authors' study population. This suggests that the current recommendations for universal vitamin D supplementation of breast-fed infants throughout the United States may need to be revised.

A treatment trial of vitamin D supplementation in breast-fed infants: universal supplementation is not necessary for rickets prevention in Southern Louisiana.

We synthesized fusion proteins of parathyroid hormone (PTH) (1-33) and the collagen binding domain of ColH (CBD) and tested them for anabolic bone activity in mice. Two fusion proteins were synthesized, linking the carboxy terminus of PTH(1-33) either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain (PTH-PKD-CBD). Both PTH-CBD and PTH-PKD-CBD increased cAMP accumulation in cells stably transfected with the PTH/PTHrP receptor, and both peptides bound to type 1 collagen in flow-through assays. Distribution studies indicated that the PTH-CBD was concentrated in the bone and skin, tissues with abundant collagen and blood flow. Administration of 320 μg/kg PTH-CBD either weekly (for 8 weeks) or monthly (for 6 months) to 7-week-old C57BL/6J mice resulted in a sustained increase in bone mineral density (BMD) (15% for weekly studies, 13% for monthly studies; P < 0.05). PTH-PKD-CBD showed only 5% increases in BMD after weekly administration, and, as expected, neither weekly nor monthly PTH(1-34) affected BMD. PTH-CBD increased serum alkaline phosphatase levels. Importantly, there were no significant increases in serum calcium observed. Collectively, the data suggest that PTH-CBD has a sustained anabolic effect in bone with either weekly or monthly administration. This approach of targeted delivery of PTH to bone may show promise for the treatment of disorders of low bone mass, such as postmenopausal osteoporosis.

Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice.

Breast-feeding imparts many benefits to both mothers and infants. Because of these numerous recognized benefits, there has been an effort to increase breast-feeding rates nationwide; increasing breast-feeding rates was one of the goals of the U.S. Department of Health and Human Services' Healthy People 2010 initiative. This study examined the breast-feeding rate at the Ochsner Clinic Foundation by conducting a retrospective chart review of patients aged 0-12 months who visited any branch of the Ochsner hospital system. Our results indicate that the rate of initiation of breast-feeding for children followed at the Ochsner Clinic Foundation is 66.3%. However, as breast-feeding rates rise, there is concern that there may be a resurgence of rickets, a disease caused by the deficiency of vitamin D. Currently the American Academy of Pediatrics recommends that exclusively breast-fed infants receive vitamin D supplementation starting within the first 2 months of life. We have therefore initiated a clinical trial to determine whether vitamin D supplementation is required to prevent rickets in breast-fed children. In the course of conducting this study, we have gathered additional data regarding the patterns of breast-feeding and of vitamin D supplementation for babies born at Ochsner.

Breast-feeding and Vitamin D Supplementation Rates in the Ochsner Health System.

Parathyroid hormone (PTH) agonists and antagonists have been shown to improve hair growth after chemotherapy; however, rapid clearance and systemic side-effects complicate their usage. To facilitate delivery and retention to skin, we fused PTH agonists and antagonists to the collagen binding domain (CBD) of Clostridium histolyticum collagenase. in-vitro studies showed that the agonist fusion protein, PTH-CBD, bound collagen and activated the PTH/parathyroid hormone-related peptide receptor in SaOS-2 cells. The antagonist fusion proteins, PTH(7-33)-CBD and PTH([-1]-33)-CBD, also bound collagen and antagonized PTH(1-34) effect in SaOS-2 cells; however, PTH(7-33)-CBD had lower intrinsic activity. Distribution studies confirmed uptake of PTH-CBD to the skin at 1 and 12 hr after subcutaneous injection. We assessed in vivo efficacy of PTH-CBD and PTH(7-33)-CBD in C57BL/6J mice. Animals were depilated to synchronize the hair follicles; treated on Day 7 with agonist, antagonist, or vehicle; treated on Day 9 with cyclophosphamide (150 mg/kg i.p.) or vehicle; and sacrificed on Day 39. Normal mice (no chemo and no treatment) showed rapid regrowth of hair and normal histology. Chemo+Vehicle mice showed reduced hair regrowth and decreased pigmentation; histology revealed reduced number and dystrophic anagen/catagen follicles. Chemo+Antagonist mice were grossly and histologically indistinguishable from Chemo+Vehicle mice. Chemo+Agonist mice showed more rapid regrowth and repigmentation of hair; histologically, there was a normal number of hair follicles, most of which were in the anagen phase. Overall, the agonist PTH-CBD had prominent effects in reducing chemotherapy-induced damage of hair follicles and may show promise as a therapy for chemotherapy-induced alopecia.

Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain.

Vitamin D is critical in bone and mineral homeostasis, particularly in the prevention of rickets in children. Levels of vitamin D in cord blood were measured in a population from New Orleans as an index of maternal vitamin D status at the time of delivery. Cord blood samples from infants born during the summer and winter showed lower 25-hydroxyvitamin D levels compared with those from infants born during fall and spring, indicating an unusual pattern of seasonality where vitamin D levels were among the lowest in the season with the greatest sunlight. It is important to establish screening and supplementation guidelines based on observed regional trends and risk factors, in addition to considerations based on global recommendations.

Reversed seasonal variation in maternal vitamin D levels in southern Louisiana.

Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1-33) to a collagen binding domain (PTH-CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH-CBD (320 μg/kg). PTH-CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH-CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH-CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH-CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH-CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.

A single injection of the anabolic bone agent, parathyroid hormone-collagen binding domain (PTH-CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice.

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. This study compared the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50-150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6-12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.

Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.

Alopecia areata is a common form of hair loss in which autoimmune-mediated destruction of hair follicles causes patchy hair loss, for which there is no adequate therapy. Parathyroid hormone (PTH) induces the hair cycle and promotes hair growth. PTH-CBD is a fusion protein of PTH and a bacterial collagen-binding domain (CBD), leading to targeted delivery to and retention in the skin collagen. We tested the effects of a single dose of PTH-CBD (low or high dose) on an animal model for alopecia areata, the C3H/HeJ engrafted mouse. In all the treated animals, there was a rapid (1-4 days) increase in hair growth, with sustained effects observed over a 2-month period (7/10 total treated mice<40% hair loss based on gray scale analysis, vs. 2/5 in vehicle control animals). Histological examination revealed massive stimulation of anagen VI hair follicles in treated animals despite an ongoing immune response. PTH-CBD thus shows promise as a therapy for alopecia areata, likely in conjunction with a mild immune suppressant, such as hydrocortisone cream.

Therapy for alopecia areata in mice using parathyroid hormone agonists and antagonists, linked to a collagen-binding domain.

The pharmacokinetics of a hybrid peptide consisting of the N-terminal biologically active region of human parathyroid hormone (PTH) linked to a collagen-binding domain (CBD) were evaluated in female Sprague-Dawley rats. The peptide, PTH-CBD, consists of the first 33 amino acids of PTH linked as an extension of the amino acid chain to the CBD peptide derived from ColH collagenase of Clostridium histolyticum. Serum concentrations arising from single dose administration by the subcutaneous and intravenous routes were compared with those measured following route-specific mole equivalent doses of PTH(1-34). Population-based modeling demonstrated similar systemic absorption kinetics and bioavailability for both peptides. Exposure to PTH-CBD was sixfold higher because of a systemic clearance of approximately 20% relative to PTH(1-34); however, these kinetics were consistent with more than 95% of a dose being eliminated from serum within 24 h. Results obtained support continued investigation of PTH-CBD as a bone-targeted anabolic agent for the treatment of postmenopausal osteoporosis.

Pharmacokinetics in rats of a long-acting human parathyroid hormone-collagen binding domain peptide construct.

Chemotherapy-induced alopecia is a major source of psychological stress in patients undergoing cancer chemotherapy, and it can influence treatment decisions. Although there is currently no therapy for alopecia, a fusion protein of parathyroid hormone and collagen binding domain (PTH-CBD) has shown promise in animal models. The aim of this study was to determine whether there are dose-dependent effects of PTH-CBD on chemotherapy-induced alopecia in a mouse model. C57BL/6J mice were waxed to synchronize hair follicles; treated on day 7 with vehicle or PTH-CBD (100, 320, and 1000 mcg/kg subcutaneous injection); and treated on day 9 with vehicle or cyclophosphamide (150 mg/kg intraperitoneally). Mice were photographed every 3-4 days and killed on day 63 for histological analysis. Photographs were quantified by gray scale analysis to assess hair content. Mice not receiving chemotherapy showed regrowth of hair 2 weeks after waxing and normal histology after 2 months. Mice receiving chemotherapy alone showed marked hair loss after chemotherapy, which was sustained for 10 days and was followed by rapid regrowth of a normal coat. Histological analysis revealed rapid cycling dystrophic anagen/catagen follicles. Animals receiving chemotherapy and PTH-CBD showed decreased hair loss and more rapid regrowth of hair than that seen with chemotherapy alone (increased hair growth by gray scale analysis, P<0.05), and the effects were dose dependent. Histologically, hair follicles in animals receiving the highest dose of PTH-CBD were in a quiescent phase, similar to that in mice that did not receive chemotherapy. Single-dose subcutaneous administration of PTH-CBD showed dose-dependent effects in minimizing hair loss and speeding up recovery from chemotherapy-induced alopecia.

Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner.

Tulasi Ponnapakkam

Pediatric Endocrinology

Children's Hospital at Montefiore

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Tulasi Ponnapakkam

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Pediatric Endocrinology

Children's Hospital at Montefiore

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Pediatric Endocrinology