Masson-Goldner Trichrome Research Articles

Mode of Growth Hormone Action in Osteoblasts

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.

Topical high-molecular-weight hyaluronan and a roofing barrier sheet equally inhibit postlaminectomy fibrosis

Topical high-molecular-weight hyaluronan and a roofing barrier sheet equally inhibit postlaminectomy fibrosis

Prevention of epidural fibrosis after laminectomy: 3-week and 8-week study using a barrier sheet and hyaluronan gel

Purpose of study: Epidural fibrosis contributes to failed back surgeries. Previously, many methods were used to reduce postlaminectomy scar with mixed results. The purpose of this study is to: 1) compare the effect of a thin barrier (Macropore) sheet and a hyaluronan (HA) gel using a control laminectomy model; 2) to examine the incisional wound integrity after treatment; and 3) to compare long-term and short-term results.Methods used: Bilateral laminectomies (L5 and L6) and a disc injury (L5–L6) were performed in 80 male adult Sprague-Dawley rats. There were four groups: 1) normal controls without surgery; 2) laminectomy without treatment; 3) HA gel and 4) a Macropore barrier sheet. The animals were sacrificed at 3 and 8 weeks for analysis. Sixty-six specimens were analyzed biochemically to determine the amount of total collagen from hydroxyproline content in the dura and the L5 nerve roots. Fourteen specimens were prepared and stained with Masson-Goldner Trichrome to assess collagen distribution. Twenty-seven healing surgical incisions were analyzed by uniaxial tensile testing to determine ultimate force, strain and prefailure stiffness. Statistics were performed using analysis of variance.of findings: Gross appearance and histology show that the untreated laminectomy scar becomes more adherent to the dorsum of the dura mater with time, whereas the barrier sheet and the HA gel prevented scar attachment to the dural sleeve in both the 3-week and 8-week study. The treatment groups showed significant (p<.0001 to .03) reduction of total collagen content in the laminectomy site in both time periods. However, the 8-week untreated group showed a significant (p=.0216) increase in total collagen content compared with the 3-week results. The HA and the Macropore barrier sheet each appeared to reduce scar around the nerve roots on histological sections, and the nerve roots were not as adherent to the adjacent pedicle grossly. However, biochemically the collagen content was not significantly different. The mechanical properties of incisional wounds increased significantly (p<.0001) between 3 weeks and 8 weeks. The ultimate strength, stress, strain and stiffness of untreated, HA and Macropore wounds were similar within each time point.Relationship between findings and existing knowledge: Wound healing experiments using HA are based on the concept of fetal healing. Fetal wounds heal without scar during the first two trimesters of gestation. Application of HA has been shown to decrease cutaneous scar formation in various models. Thus, HA represents a possible candidate for reduction of postlaminectomy scar formation. The barrier sheet, by walling off the overlying muscle may be effective in reducing cellular trafficking and vascular ingrowth into postlaminectomy defect. Because the barrier is a confined substrate, it should not interfere with normal posterior incisional wound healing. The current study confirmed the hypothesis that postlaminectomy scar formation can be attenuated by application of HA or placement of a particular barrier sheet. Furthermore, we show the antifibrotic agent used did not interfere with the incisional wound healing.Overall significance of findings: HA and Macropore barrier sheet treatment significantly reduced postlaminectomy peridural scar formation in this model without affecting the integrity of incisional wound healing. Furthermore, our results suggest untreated postlaminectomy scar is maturing with time, as indicated by increase collagen content and improvement of incisional mechanical properties.Disclosures: Device or drug: Healon. Status: approved. Device or drug: Macropore (70:30 absorbable poly L-lactide-co-D and Poly L-lactide). Status: approved.Conflict of interest: Jennifer Massie, grant research support: VA Merit Grant.

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy

Purpose: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation.Setting: Medical Laser Center Lübeck, Germany.Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 μm) and a pulsed chromium-thulium-holmium:YAG laser (2.1 μm) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbium:YSGG laser (2.70 μm), a free-running erbium:YAG laser (2.94 μm), and an argon-fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson's, and Masson-Goldner's trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius-red staining. Sirius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence.Results: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius-red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius-red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence.Conclusion: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

A synthetic polypentapeptide of elastin for initiating calcification.

A polypentapeptide (PPP) of tropoelastin having a repeating amino acid sequence of (Val-Pro-Gly-Val-Gly)n was evaluated for its potential to initiate in vivo calcification and to enhance bone formation in nonhealing calvarial wounds (8.0 mm) in 396 adult Walter Reed rats. There were four configurations of the PPP (molecular weight range of 50-100K dalton) consisting of 1-dry PPP; 2-coacervate PPP; 3-gamma irradiated, cross-linked PPP; 4-calcified, gamma irradiated, cross-linked PPP. These four iterations plus a control group of animals constituted the five treatment classes that were evaluated at days 1, 3, 7, 21, 42, and 147. Seventy two rats were used for each treatment and 36 rats for the control. Following euthanatization, specimens were placed into 70% ethanol, embedded in polymethyl methacrylate, sectioned at 3.5 micrometers, and alternating sections were stained with Masson-Goldner trichrome and von Kossa stains. Histomorphometric analysis was accomplished using a Zeiss Universal microscope (250 X) and Videoplan Image Analysis System to evaluate five random histologic fields from margin to margin of the craniotomy. Trabecular bony volume and area of calcification islands were quantitated. A Student's t test for unpaired data to determine treatment differences (within the same temporal groups) revealed that there was no significant difference between treatments and control for trabecular bony volume; however, there was a significant difference between experimentals and control for calcification islands (P less than 0.05) such that calcifications islands for the experimentals were greater than the control. There was not a significant difference between experimental treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and histochemical studies on the cardiac conduction system of the dog.

The conduction system of the dog heart was studied morphologically and histochemically.Seventy five fresh hearts of dogs of both sexes were examined partly in sections (hematoxlyin-eosin, azan, Masson-Goldner trichrome staining and periodic acid-Schiff reaction) and partly in macroscopic preparations treated with iodine reaction and modified PAS reaction (OTSUKA and HARA 1965). The following findings were obtained.1. The sinus node is composed of an irregular network of muscle fibers finer than the ordinary auricular muscle. They are richly ramified and contain a few myofibrils and abundant sarcoplasma. Connective tissue fibers intervene among the nodal fibers. The sinus node has no clear-cut boundary. Each nodal fiber shows smooth transition to an ordinary muscle fiber at the periphery of the node. A specialized muscular pathway connecting the sinus node with the atrioventricular node is not recognized.2. The atrioventricular node consists of the posterior and anterior portions. The former is close to coronary sinus-orifice and the latter continues to the atrioventricular bundle. Histologically, the former resembles the sinus node and the latter the atrioventricular bundle. Between the two, there is no boundary. The atrioventricular node is found in the connective tissue of the anulus fibrosus and separated from the ordinary heart muscle. In the posterior part of the atrioventricular node, there is a muscular pathway connecting the right auricle with the atrioventricular node. This pathway consists not of specialized, but of ordinary auricular muscle fibers. There is no other “bypass tracts” (JAMES 1963) between the atrium and the atrioventricular node.3. The atrioventricular bundle is composed of thick fibers with few ramifications. They are rich in sarcoplasma and glycogen and poor in myofibrils when compared with ordinary cardiac muscle fibers. This specialized tissue resembles that of ungulates. These fibers first run forming a single bundle, then fork into two main bundles, right and left.4. The right main bundle courses in the subendocardium as a single cord, drawing an arch (convex to pulmonary conus), without branching. Ariving at the anterior papillary muscle, it issues branches for 3 areas: for the conus pulmonalis, for the free wall, and for the septum.5. The left main bundle becomes visible beneath the aortic valve as a wide bundle and spreads over the subendocardium as a fimbria, giving three groups of branches. Branches of the first group run toward the anterior papillry muscle via pseudotendons. Those of the second group reach the posterior papillary muscle via pseudotendons and give rise to a very thick distribution of Purkinje fibers to the left ventricular free wall. The third group of the peripheral branches spreads directly over the septum without passing pseudotendons.6. The network of Purkinje fibers in the subendocardium is the thickest on the free wall of the both ventricles and the thinnest on the right side of the interventricular septum.7. There is much glycogen throughout the conduction system, especially in the atrioventricular bundle and Purkinje fibers. The glycogen is less soluble in water compared with that of the ordinary heart muscle. Physiological meaning of this glycogen is not yet clear.