Irradiated Limb Research Articles

Contrast-Enhanced Ultrasound to Detect Early Microvascular Changes in Skeletal Muscle after High-Dose Radiation Treatment.

The biological response of normal tissue to high-dose radiation treatment remains poorly understood. Alterations to the microenvironment, specifically the microvasculature, have been implicated as a significant contributor to tumoral cytotoxicity. We used contrast-enhanced ultrasound (CEU) perfusion imaging, which is uniquely suited to assess functional status of the microcirculation, to measure microvascular blood flow after high-dose irradiation to normal skeletal muscle tissue in a murine model. Proximal hindlimbs of wild-type C57Bl/6 mice were irradiated with a single fraction using 6 MV photons, 1 cm bolus and a dynamic wedge. Quantitative perfusion CEU imaging of the skeletal muscle was performed at days 1 and 8 postirradiation in three different regions of interest (ROIs): 1. 15 Gy external-beam irradiated leg; 2. 12 Gy irradiated 5 mm proximal area; 3. single ROI in the nonirradiated contralateral (CL) hindlimb. Perfusion imaging was also performed in the hindlimb of nonirradiated mice. CEU time-intensity data were analyzed to measure microvascular blood flow (MBF, also referred to as perfusion), and its parametric components of microvascular flux rate and functional microvascular blood volume (MBV). Plasma measurements of two potent vasoconstrictors, endothelin-1 and angiotensin II, were also performed to assess systemic response. CEU perfusion imaging values for the 12 and 15 Gy irradiated limb regions were pooled. At day 1, MBF in the irradiated limb was significantly lower than in the CL limb (P = 0.016) but quite similar to the nonirradiated mice. At day 8, both limbs of irradiated mice exhibited a trend towards lower MBF than the limbs of nonirradiated mice (28% decrease in mean MBF, P = 0.149 for CL; 39% decrease, P = 0.065 for irradiated limb). Compared to nonirradiated animals, the reduction in perfusion in irradiated limbs at day 8 may have been more influenced by the microvascular flux rate (25% decrease in the mean, P = 0.079) than the MBV (12% decrease in the mean, P = 0.328). Examination of vasoactive compounds revealed that the average plasma concentration for endothelin-1 at day 8 postirradiation was significantly higher in 14 irradiated animals than in 4 nonirradiated animals (3.07 pg/ ml vs. 2.51 pg/ml; P = 0.011). Up to day 8 after high-dose irradiation, flow deficits in irradiated muscle appear to be a consequence of increased vascular resistance more so than loss or functional de-recruitment of microvascular units.

AN IN VIVO STUDY OF THE EFFECTS OF IONIZING RADIATION ON TISSUES BY LASER FLUORESCENCE SPECTROSCOPY

Актуальность. Лазерная флуоресцентная спектроскопия (ЛФС) широко используется в различных медицинских направлениях, наиболее известное из которых – онкология. В основном ЛФС применяют для in vivo диагностики опухолей. Последние исследования показали, что ЛФС можно использовать для диагностики локального воспаления, инициированного термическим или механическим воздействием. Представляет интерес, возможно ли оценить методом ЛФС поражение мягких биологических тканей, вызванное лучевым воздействием. Цель – исследование динамики флуоресценции экзогенного фотосенсибилизатора в области лучевого поражения методом ЛФС in vivo. Материал и методы. В исследовании участвовали мыши линии SHK (n=12), правая задняя конечность которых подвергалась облучению на гамма-терапевтическом аппарате РОКУС-АМ (источник 60Co, доза 15 Гр). Перед облучением всем животным внутрибрюшинно был введен фотосенсибилизатор Фотосенс из расчета 2,5 мг/кг. Измерения флуоресценции проводили на комплексе многофункциональной лазер- ной диагностики «ЛАКК-М» в течение 21 суток, длина волны возбуждения флуоресценции – 635 нм. Параллельно на 7- и 21-е сутки проводили гистологическое исследование области лучевого поражения и исследование лейкоцитарной формулы крови экспериментальных животных. Результаты. Метод ЛФС выявил увеличение накопления фотосенсибилизатора в области поражения по сравнению с интактной симметричной областью, что приводило к повышению интенсивности сигнала флуоресценции в облученной конечности. Динамика сигнала флуоресценции пораженной области имеет два характерных максимума – на 3-и и 14-е сутки, что, возможно, отражает динамику развития локального лучевого поражения. Заключение. Применение метода ЛФС с использованием экзогенного фотосенсибилизатора имеет потенциал для персонализированной оценки лучевых реакций в радиологии.

Radioprotective Properties of Indralin in Combination with Monizol in the Treatment of Local Acute and Delayed Radiation Injuries Caused by Local Skin γ-Irradiation.

Female rats were exposed to local γ-irradiation of the right hindpaw in doses of 30-50 Gy at 131-154 sGy/min dose rate. Radioprotector indralin was administered per os 15 min prior to irradiation, monizol was injected intraperitoneally 5 min after irradiation. Indralin showed marked radioprotective properties both for acute and delayed symptoms of local radiation injuries. In combination with monizol, radioprotective effect of indralin was potentiated to dose reduction factor of 1.4-1.5 both for radiation burn severity reduction and for restriction of postradiational contracture development and amputation of the irradiated limb.

Delayed Marrow Infusion in Mice Enhances Hematopoietic and Osteopoietic Engraftment by Facilitating Transient Expansion of the Osteoblastic Niche

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24 hours compared with 6 hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.

Physeal Bystander Effects in Rhabdomyosarcoma Radiotherapy: Experiments in a New Xenograft Model

Radiotherapy used in the treatment of pediatric musculoskeletal sarcomas may result in crippling defects of skeletal growth. Several radioprotective strategies have shown potential for preserving function of the irradiated epiphysis but have not been evaluated in a tumor-bearing animal model. We developed two bioluminescent human rhabdomyosarcoma cell lines that were used to establish xenograft tumors in skeletally immature mice. Bioluminescence imaging and radiography allowed serial evaluation of tumor growth and tibial elongation following localized radiotherapy. High-dose (10 Gy) radiotherapy significantly reduced tumor growth velocity and prolonged the median survival of tumor-bearing mice but also resulted in a significant 3.3% shortening of the irradiated limb. Exposure to a lower, 2 Gy dose resulted in 4.1% decrease in limb length but did not extend survival. This new model provides a clinically relevant means to test the efficacy and safety of novel radioprotectant and radiorecovery strategies for use in this context.

Effects of Sublethal Irradiation on Patterns of Engraftment after Murine Bone Marrow Transplantation

Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of various immunosuppressive, yet nonmyeloablative preparative regimens that often include low-dose irradiation. To determine the effects of low-dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard endpoint flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10-90) post-BMT periods. To exclude the contribution of irradiation on reducing immunologic rejection, severely immune-deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sublethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrowcompared with nonirradiated recipients (P < .05). Bioluminescence imaging demonstrated that theeffect of TBI (P < .001) on chimerism occurred only after the first 7 days post-BMT. Flow cytometric analysison day 3 showed no increase in the number of donor cells in irradiated bone marrow, confirming that sublethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intrafemoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with i.v. administration (P< .05). Overall, the combination of TBI and i.v. administration of donor cellsprovided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sublethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.

DNA Double-Strand Breaks after Percutaneous Transluminal Angioplasty

To determine exemplarily the amount of DNA damage and the repair kinetics after interventional radiologic procedures by using visualization of foci of the phosphorylated form of the H2AX histone variant (gammaH2AX) to quantify DNA double-strand breaks (DSBs) at percutaneous transluminal angioplasty (PTA) of the lower limb arteries. After local ethics committee approval and written informed consent were obtained, five patients (two women, three men; mean age, 64.4 years; age range, 45-76 years) scheduled for computed tomography (CT) and 20 patients (six women, 14 men; mean age, 68.5 years; age range, 53-85 years) scheduled for PTA of lower limb arteries were prospectively entered into the study. Blood samples were taken before the first exposure to ionizing radiation and 5 minutes, 1 hour, 6 hours, and 24 hours after the last exposure. Additional samples were taken from the irradiated limb (femoral vein) of three patients who underwent PTA--before the first radiation exposure, 5 and 10 minutes after the first exposure, and 5 minutes after the last exposure. Lymphocytes were isolated, fixed, and stained with anti-gammaH2AX antibody, and gammaH2AX focus yields were determined with fluorescence microscopy. Data were analyzed with linear regression and two-sample F tests. Mean increase in number of gammaH2AX foci after CT (7.78 per 1 Gy x cm) depended linearly on dose-length product (r = 0.997). Number of foci reached background levels within 24 hours. Mean numbers of gammaH2AX foci per cell increased by factors of 4.08-20.67 in blood samples taken 5 minutes after PTA compared with mean numbers of foci before PTA. Mean radiation dose increase, 6.56/(10 Gy x cm(2)), depended linearly on dose-area product (r = 0.993). Maximal focus yield in cells taken directly from the irradiated limb was higher than that in cells from the systemic circulation (by mean factor of 1.46). Data showed compromised DSB repair capacity after PTA (P < .05). Mean number of foci at 24 hours (0.07 focus per cell) was significantly higher than mean number of foci in cell background (0.04 focus per cell, P < .05). GammaH2AX focus formation can be used to determine in vivo induction of DNA DSBs after PTA. DSB repair capacity is compromised in patients who undergo PTA of lower limb arteries.

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

Radiation therapy encompassing an active epiphysis can negatively impact the potential for bone growth by disrupting cell-cycle progression and accelerating apoptosis and terminal differentiation in physeal chondrocytes. Despite functional derangement following radiation exposure, the irradiated growth plate retains a capacity for regeneration and recovery of growth. The purpose of this study was to characterize the initial sequence of events leading to functional growth recovery in irradiated weanling rat growth plates. We hypothesized that growth in an irradiated epiphysis would be partially restored due to the expansion of chondrocytic clones. Stereological histomorphometry was used to compare chondrocytic cell and matrix turnover between the first and second week following irradiation, and to determine the relative contribution of each of the cellular and extracellular matrix (ECM) compartments to growth. We found that restoration of growth in the irradiated limb was strongly associated with the proliferative activity and production of ECM by these chondrocytic clones, as they expand in average volume, but not in numerical density. We conclude that chondrocytes forming expansive clones and exhibiting increased mitotic and matrix synthesis activity initiate the early restoration of function in the irradiated growth plate, and would be a logical target for strategies to restore full growth potential.

Radiomodifying responses of vitamin E and epigallocatechin gallate supplementation during irradiation of implanted tumors

9601 Background: Dietary antioxidants may play an antagonistic role during radiation treatment (RT). Antioxidants such as vitamins C and E show radioprotective effects on normal cells in vitro but limited evidence in this regard is available in healthy tissue or tumor cells in vivo. Methods: Syngeneic, murine breast carcinoma (MCa-4) cells were implanted in hind legs of C3H mice. Vitamin E (VE) or epigallocatechin gallate (EGCG, derived from green tea extract) were fed at 500 and 658 mg/kg diet, respectively, for 4 wk until tumors reached 8 mm. Three tumor control dose 50 (TCD50) assays (N=48 mice/assay) were performed to quantify the radiomodifying effect of the antioxidants. Tumor growth data were collected prior to and after RT. Tissue concentrations of VE and EGCG were analyzed by HPLC. Tissue reactions were recorded for 90 d following RT. Angiogenesis specific gene arrays were used to analyze expression in tumor and cultured tumor cells. Results: A 4% increase in TCD50 was observed in mice fed either VE or EGCG supplemented diets relative to the control diet, though these changes were not statistically significant (P=0.30 and 0.20, respectively). The gamma-50 (slope) was 6.2 for control and 3.5 for both experimental arms (P=0.20). Prior to RT, tumor growth was 10% slower in the EGCG group compared to controls (P=0.002). VE concentration in the tumor was twice that of surrounding muscle (382 vs. 189 pmole/mg) but only slightly (4%) slowed tumor growth (P=0.24) prior to RT. There were fewer RT-related soft-tissue reactions (loss of irradiated limb below the tumor) in the EGCG (9.8%) and VE (7.1%) groups compared to control (23.8%) at P=0.09 and 0.04, respectively. EGCG increased VEGF and MMP gene expression in stromal tissue but decreased it in the tumor. Conclusions: TCD50 and gamma-50 were modestly increased in mice fed diets supplemented with VE or EGCG, but these changes were not statistically significant. EGCG significantly inhibited tumor growth rate. Adverse RT-related soft tissue reactions occurred less frequently with antioxidant supplementation, particularly with VE. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Harvard Medical School/Osher Institute; Goldman Philanthropic Partnerships

Vascularized bone-marrow allotransplantation in rats prolongs the survival of simultaneously grafted alloskin.

Skin allografts in rats were rejected, even if they were treated with a sub-optimal dose of FK506. However, skin allografts in rats which simultaneously received vascularized limb allografts were rejected more slowly. In this study, the authors examined the reasons why vascularized limb allografts have the ability to induce immunologic tolerance. For investigating which tissue components provide such tolerance, they compared the mean survival times (MST) of skin allografts on the backs of rats (used as an indicator of rejection), which were simultaneously engrafted with vascularized skin allografts, muscle allografts, joint allografts, and limb allografts. In the cases of limb allografts (MST=32.1 days) (these allografts contain bone-marrow cells), the skin allografts survived longer than vascularized skin allografts (MST=23.6 days) or muscle allografts (MST=22.6 days). For ascertaining whether the ability to induce immunologic tolerance of limb allografts was due to bone-marrow cells or not, the authors compared the MST of skin allografts in rats that were simultaneously engrafted with non-irradiated limb allografts, irradiated limb allografts, and bone-marrow cells. The allografted skin in the rats that received non-irradiated limbs (MST=32.1 days) survived longer than irradiated limb allografts (MST=20.3 days) and bone-marrow cells (MST=18.4 days). Limb allografts are, as it were, vascularized bone-marrow allografts. These results suggested that vascularized bone-marrow allografts can facilitate the induction of immunologic tolerance.

Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers.

Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.

Sparing Radiation-Induced Damage to the Physis by Radioprotectant Drugs: Laboratory Analysis in a Rat Model

The radioprotectant compound amifostine (S-2{3-aminopropylamino}-ethylphosphorothioc acid), administered prior to radiotherapy, has been demonstrated to provide differential protection of normal cells from the damaging effects of ionizing radiation. The aim of this pilot was to determine if amifostine could preserve the integrity of, or minimize the damage to, the physis during exposure to radiation in an animal model. Thirty weanling Sprague-Dawley rats were randomized into five groups of six animals each. Groups 1 and 2 received a single exposure to radiation consisting of 12.5 and 17.5 Gy, respectively. Groups 3 and 4 received similar exposures of 12.5 and 17.5 Gy, respectively, but with prior administration of amifostine at 100 mg/kg. Group 5 (control) received neither radiation nor amifostine. At 6 weeks, femoral and tibial lengths were measured in treated and untreated hindlimbs and compared with the baseline lengths to calculate growth. Concordant with previous reports in the literature, the radiation doses of 12.5 and 17.5 Gy reduced net femoral growth in length by a mean of 23% (range = 12–33%, SD = 7.41) and 59% (range = 54–64%, SD = 4.45), respectively, in the irradiated limb. Amifostine reduced anticipated growth loss normally resulting from a single 12.5-Gy radiation dose by 48.9% in the femur, 13.1% in the tibia, and 27.6% overall in the total limb (p ⩽ 0.05). Similarly, anticipated growth loss from a single 17.5-Gy radiation dose was reduced by 30.8% in the femur, 20.3% in the tibia, and 25.7% overall in the total limb (p ⩽ 0.05). Amifostine administered prior to clinically relevant radiation exposures significantly reduced the amount of anticipated growth arrest in our animal model.

Sparing radiation-induced damage to the physis by radioprotectant drugs: laboratory analysis in a rat model.

The radioprotectant compound amifostine (S-2[3-aminopropylamino]-ethylphosphorothioic acid), administered prior to radiotherapy, has been demonstrated to provide differential protection of normal cells from the damaging effects of ionizing radiation. The aim of this pilot was to determine if amifostine could preserve the integrity of, or minimize the damage to, the physis during exposure to radiation in an animal model. Thirty weanling Sprague-Dawley rats were randomized into five groups of six animals each. Groups 1 and 2 received a single exposure to radiation consisting of 12.5 and 17.5 Gy, respectively. Groups 3 and 4 received similar exposures of 12.5 and 17.5 Gy, respectively, but with prior administration of amifostine at 100 mg/kg. Group 5 (control) received neither radiation nor amifostine. At 6 weeks, femoral and tibial lengths were measured in treated and untreated hindlimbs and compared with the baseline lengths to calculate growth. Concordant with previous reports in the literature, the radiation doses of 12.5 and 17.5 Gy reduced net femoral growth in length by a mean of 23% (range = 12-33%, SD = 7.41) and 59% (range = 54-64%, SD = 4.45), respectively, in the irradiated limb. Amifostine reduced anticipated growth loss normally resulting from a single 12.5-Gy radiation dose by 48.9% in the femur, 13.1% in the tibia, and 27.6% overall in the total limb (p < or = 0.05). Similarly, anticipated growth loss from a single 17.5-Gy radiation dose was reduced by 30.8% in the femur, 20.3% in the tibia, and 25.7% overall in the total limb (p < or = 0.05). Amifostine administered prior to clinically relevant radiation exposures significantly reduced the amount of anticipated growth arrest in our animal model.

Effects of radiation on fixation of non-cemented porous-coated implants in a canine model.

A non-weight-bearing porous-coated rod was implanted bilaterally in the proximal part of the humerus in thirty-five adult male mongrel dogs. In all of the animals, one limb was treated with radiation and the opposite limb served as the control. In twenty-one animals, the dose was 1000 centigrays (rads) and in fourteen, it was 500 centigrays. The strength of fixation and the volume fraction of ingrowth of bone were determined two, four, and eight weeks after the operation in the group that received 1000 centigrays and two and four weeks after the operation in the group that received 500 centigrays. Treatment with 500 centigrays had no significant effect on the strength of fixation or the amount of ingrowth of bone. In contrast, at two weeks, treatment with 1000 centigrays had reduced the strength of fixation to 50 per cent of the control value (p less than 0.01), although, at four and eight weeks, the strength of fixation was not significantly different than that in the control limb. The amount of ingrowth of bone in the irradiated limb was significantly reduced at two weeks (30 per cent of the control value) (p less than 0.01), four weeks (70 per cent of the control value) (p less than 0.05), and eight weeks (56 per cent of the control value) (p less than 0.05).

The Effect of Radiation Therapy on the Fixation Strength of an Experimental Porous-Coated Implant in Dogs

Radiation therapy prevents heterotopic ossification after cemented total hip arthroplasty, but its effect on the fixation of porous-coated devices is not known. The purpose of this study was to determine the effect of irradiation on the pullout strength of a porous implant in the canine model. A custom-designed titanium fiber metal-coated cylinder was implanted into the distal femurs of 12 dogs. One femur of each dog was irradiated with 10 Gy, given in four 2.5-Gy daily fractions beginning one day after surgery. Serial bone scans were performed at one, two, four, and six weeks postoperatively. At six weeks, the femurs were harvested, and eight were randomly selected for mechanical testing. Scintigraphic evaluation showed a significant reduction in uptake at one and two weeks in the irradiated limb, but this had returned to normal at four and six weeks. The failure to pullout was significantly less in the irradiated limb compared with the control limb six weeks postoperatively. In this canine model, the pullout strength of the titanium fiber implant was significantly reduced by a dose of radiation commonly used in the prevention of heterotopic ossification. If irradiation is chosen for prophylaxis, either the porous implant should be adequately shielded or a cemented implant should be utilized.

Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb.

A set of tendons, aponeurotic sheets and retinaculae, which transduce muscle action from proximal limb levels to flexion and extension of the digits, is found in limbs of many vertebrates. This set of structures, here termed the digit tendon complex, is described for the axolotl forelimb. We show that the complex forms autonomously in muscleless axolotl limb regenerates produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a year after amputation. The pattern of other connective tissue structures, including the skeleton, is also normal. Fibroblast condensations that may represent sets of these cells normally associated with muscles in the extensor and flexor compartments of the carpal region also form in muscleless limbs. The results are discussed in terms of the importance of the dermis in pattern regulation, selforganization of connective tissues in general and autonomous development of the digit tendon complex in particular.

Replacement of irradiated epidermis by migration of non-irradiated epidermis in the newt limb: the necessity of healthy epidermis for regeneration

An X-irradiated newt limb is able to regenerate if non-irradiated skin as well as non-irradiated muscle is transplanted to the stump. Non-irradiated epidermis is brought to the stump with a skin graft but not with a muscle graft. In order to know whether limb regeneration required healthy epidermis or not, a triploid skin cuff was set at the most proximal level of an irradiated limb and muscle was transplanted to the level of the midforearm. The forearm was then amputated through the muscle graft. A cytophotometrical analysis of DNA content of the epidermis cell nuclei sampled from the skin of the regenerate was undertaken to detect a migration of triploid epidermal cells. The result was a complete replacement of diploid irradiated epidermis by triploid epidermis, during the six weeks necessary for regeneration. Another investigation consisted of detecting a possible migration of non-irradiated triploid epidermis along an irradiated limb which had not been amputated. Healthy epidermis was found to migrate distally and replace irradiated epidermis in three weeks. Previous experiments involving transplantation of a non-irradiated skin cuff or muscle to an irradiated limb stump were carried out again but on animals which had been entirely irradiated to prevent any extra healthy epidermis cells from contaminating the regenerating limb epidermis. A regenerate developed from the skin graft but not from muscle graft. It is concluded that healthy epidermis must be present on the limb stump to permit the blastema to develop.

Korrelation zwischen radiologischen, szintigraphischen und histologischen Veränderungen im Knochen beim Kaninchen nach Bestrahlung mit Einzel- und fraktionierter Dosis*

The left hind legs of eight rabbits were irradiated with 1.750 rad in a single dose or with 4,650 rad divided over a period of three weeks. These animals, as well as four who had not been irradiated, were examined periodically radiologically and with 99m Technetium pyrophosphate scintigrams during one year. No difference could be detected between the results of the single and fractionated doses. Scintigraphically there was a biphasic increase in uptake, the first peak co-inciding with the irradiation and of vascular origin, whereas the second peak occurred at four months and was due to a change in bone metabolism. At the end of a year uptake in the irradiated limb was slightly reduced. The earliest radiological changes were found after six months, at the end of the second scintigraphic peak; they consisted of coarsening and blurring of the trabeculae, nonhomogeneous spotty mineralisation and endosteal scalloping. Six animals developed a radiation-induced bone sarcoma, first demonstrated either by scintigraphy or radiology depending on its histology. The value of combining radiological and scintigraphic examinations for the early detection of post-radiation abnormalities is stressed.

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10 6 cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [ 3H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.

Restoration of osteogenic properties of bone marrow after irradiation in heterotopic transplantation experiments

After subcapsular transplantation of tibial bone marrow, locally irradiated in a dose of 2000 R, into the kidney of mice of the same line 0–4 months after irradiation, resporption always took place. Meanwhile, bone marrow from the unirradiated limb of the same donors always possessed osteogenic potential. The stroma of the irradiated limb was defective and did not regenerate by repopulation of the stromal cells from unirradiated areas. Experiments in which mice were irradiated repeatedly and the same limb was shielded gave similar conclusions. The results of heterotopic transplantation show that stromal cells, unlike hematopoietic cells, cannot repopulate.