Biological rhythms of tissue basophils of the dura mater of rats under the effects of noise-vibration

The biorhythmologic changes of tissue basophils (mast cells) have been investigated of mater rats in frontal, temporal and occipital regions of dura mater during the noise-vibration stress. Local differences were found in biorythmological characteristics of tissue basophils before and after vibration. The investigated regions of dura mater after noise-vibration stress the magnitude of harmonics, mesor and the amplitude of fluctuations increased greatly. But, judging by their values, in the temporal region the rhythm stability was lower than that in the frontal and occipital. The tissue basophils of various regions of dura mater react in a different way to the noise-vibration stress.     

AN ELECTRON MICROSCOPE STUDY OF VITALLY STAINED SINGLE CELLS IRRADIATED WITH A RUBY LASER MICROBEAM

An electron microscope study has been made of vitally stained single cells whose cytoplasm has been subjected to a localized ruby laser microbeam. Light and moderate laser absorption (the resultant of stain concentration and laser energy density) produced restricted selective damage of mitochondria in cells stained with Janus green B; heavy laser absorption resulted in mitochondrial damage, as well as in nonselective interaction with other cell structures. With four other basic vital stains, the polysomes, ergastoplasm, mitochondria and other organelles at the irradiated site were uniformly damaged. Unstained cells showed no morphological alterations. With light primary damage (that restricted to the irradiation site), no secondary effects of the incident radiation were observed. With moderate primary damage, however, secondary damage of the mitochondria in the unirradiated cell portions was produced, which was reversible within 4 hr after irradiation. Heavy primary lesions caused severe secondary alteration of all cell structures that was irreversible and cell death occurred within 2 hr. Surviving cells examined 24 hr after light and moderate irradiation could not be distinguished from unirradiated controls. The possible mechanisms involved in the production of laser-induced cellular alterations are discussed.     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

Photodynamic action of endogenously synthesized porphyrins from aminolevulinic acid,using a new model for assaying the effectiveness of tumoral cell killing

• 1.1. The effectiveness of the photodynamic action of porphyrins, was studied by means of the tissue explant culture technique. A murine tumor tissue explant was incubated in a medium containing 0.6 mM of ALA for periods of 1 and 2 hr; total porphyrins synthesized under these conditions were of the same level as those found in our previous in vivo experiments. The explants were then irradiated for 30 min with He-Ne laser of 3.5 mW output power placed at a distance of 10 cm. Controls of non-irradiated tumor tissue slices incubated with and without ALA were performed. Immediately after irradiation, innocula of exactly 1 mm³ of the irradiated and non-irradiated tissue were subcutaneously injected under the right and left flanks of the same animal, respectively. The growth of the tumor was measured 15, 20 and 25 days after implantation.
• 2.2. Results obtained showed that the explants that were incubated for 1 hr with ALA and irradiated, reaching a concentration of 2.8 μg porphyrins/g tissue, produced a reduction of 50–70% of tumor size as compared with the non-irradiated controls incubated with ALA. Explants incubated for 2 hr, reaching a concentration of 4.6 μg porphyrins/g tissue, produced from 60% to complete lack of tumor growth. The effectiveness index (EI) of photoirradiation was calculated on the basis of the tumor growth in irradiated and non-irradiated tumors. EI was nearly 100% showing almost complete tumor cell destruction for tumor irradiated for 2 hr with 0.6 mM ALA.
• 3.3. As indicators of cell injury and subsequent death and necrosis, LDH activity in the incubation medium and intracellular potassium content were measured. Results indicated that as a consequence of irradiation of porphyrin loaded tumor explants, significant release of LDH to the medium and loss of intracellular potassium occurred. These findings show great to complete tumor destruction by combination of porphyrins endogenously formed from ALA and low irradiance with laser.

     

The effect of low-intensity laser irradiation in the blue, green, and red spectral ranges on healing of experimental skin wounds in rats

The effects of low-intensity laser irradiation in the red (632.8 nm), green (532 nm), and blue (441.2 nm) spectral ranges on wound healing has been studied in rats. The effect of the traditionally used red laser irradiation has been compared with the effect caused by laser irradiation in other spectral ranges, aiming to support the provisional hypothesis that a similar healing effect could be achieved at lower doses of wound irradiation by lasers emitting in the blue and green spectral ranges. The following parameters have been used to characterize healing of the experimental wounds: the functional activity of phagocytes in the wound exudate, which was determined from luminol-dependent chemiluminescence, the phagocyte number; the wound exudates’ antioxidant activity; and the rate of healing, which was determined as the change of the wound surface area. It was found that in all cases the laser irradiation accelerated the healing of wounds. Exposure to red laser irradiation at the dose of 1.5 J/cm²), and to blue or green laser irradiation at a dose of 0.75 J/cm² shortened the time of the wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes in irradiated groups increased by day 5 after surgery, whereas in the control group it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after surgery. Laser irradiation in the red spectral range at a dose of 1.5 J/cm² resulted in a larger increase in superoxide dismutase activity, as compared to that found after exposure to laser irradiation in the blue and green spectral ranges at a dose of 0.75 J/cm².     

Blue laser light increases perfusion of a skin flap via release of nitric oxide from hemoglobin

It has recently been shown that nitrosyl complexes of hemoglobin (NO-Hb) are sensitive to low-level blue laser irradiation, suggesting that laser irradiation can facilitate the release of biologically active nitric oxide (NO), which can affect tissue perfusion. The aim of this study was to evaluate the therapeutic value of blue laser irradiation for local tissue perfusion after surgical intervention. Blood was withdrawn from a rat, exposed to NO and infused back to the same rat or used for in vitro experiments. In vitro, an increase of NO-Hb levels (electron paramagnetic resonance spectroscopy) up to 15 microM in rat blood did not result in the release of detectable amounts of NO (NO selective electrode). Blue laser irradiation of NO-Hb in blood caused decomposition of NO-Hb complexes and release of free NO. Systemic infusion of NO-Hb in rats affected neither systemic circulation (mean arterial pressure) nor local tissue perfusion (Doppler blood flow imaging system). In contrast, a clear enhancement of local tissue perfusion was observed in epigastric flap when elevated NO-Hb levels in blood were combined with local He-Cd laser irradiation focused on the left epigastric artery. The enhancement of regional tissue perfusion was not accompanied by any detectable changes in systemic circulation. This study demonstrates that blue laser irradiation improves local tissue perfusion in a controlled manner stimulating NO release from NO-Hb complexes.     

Study of the effects of laser irradiation (540 nm) on some components of blood enzyme antioxidant system

The influence of blood laser irradiation (lambda = 540 nm) in doses range 0.3-1.2 J/cm2 on the neutrophyles superoxide-dismutase and katalase activity has been studied. Correcting effect of laser light (0.6; 1.2 J/cm2) on the functional activity of studied enzymes has been found. The high degree of correlation (r = 0.6) between neutrophil superoxide dismutase and neutrophil catalase activity of the irradiated blood has been discovered. The close correlation between components of the antioxidant system and processes of active oxygen metabolite generation has been revealed. The scheme of free radical mechanisms of the laser irradiation influence on the several parts of the enzyme antioxidant system has been suggested.     

Rejection of ticks from guinea pigs by anti-hapten-antibody-mediated degranulation of basophils at cutaneous basophil hypersensitivity sites: role of mediators other than histamine

Previous studies have established that recruitment of basophils to sites of tick feeding in guinea pigs is required to effect immune resistance. In the current study, actively sensitized guinea pigs treated three times daily with H-1 (mepyramine) and H-2 (cimetidine) histamine receptor antagonists, during the challenge tick infestation period, expressed normal resistance to Amblyomma americanum larvae. Similarly, naive guinea pigs treated with anti-histamines four times daily, beginning 7 days before transfer of immune serum and tick challenge and continuing through the tick infestation period, also expressed normal antibody-mediated resistance to A. americanum. These results indicated that histamine was not an important basophil mediator of the resistance response. Ticks allowed to feed on tissue rich in basophils that were induced by sensitization and subsequent local challenge with non-tick protein antigen, keyhole limpet hemocyanin (KLH), expressed normal yield. Ticks that fed on similar tissue rich in basophils induced by sensitization and challenge with KLH, in which the basophils expressed anti-picryl specificity due to systemic passive transfer of anti-picryl antibodies, were rejected when basophils were induced to degranulate by i.v. challenge with picryl antigen at 6 hr (29% rejection), 12 hr (18% rejection), 24 hr (22% rejection), and 48 hr (37% rejection) post-tick attachment. However, basophil degranulation at 18, 72 and 96 hr post-tick attachment had no adverse effect on tick feeding. These hosts were protected from systemic anaphylaxis by treatment with the anti-histamine mepyramine. Release of histamine occurred at tick feeding sites, but vasoactive effects were blocked by mepyramine treatment as evidenced by a lack of increased vascular permeability (bluing) at these sites compared with non-tick-infested tissues, or to cutaneous basophil hypersensitivity (CBH) sites of animals not protected with mepyramine. These results indicate that local recruitment and subsequent degranulation of basophils via immune mechanisms dependent on non-tick antigens can lead to tick rejection, and that basophil-derived mediators other than histamine are involved in this immune resistance response to A. americanum ticks. The identity of the crucial basophil mediator(s) is not known. The significant susceptibility of ticks to basophil-mediator release at 6 to 12 hr and 24 to 48 hr post-attachment coincides with the tick attaching and fast-feeding phases, respectively, suggesting that these phases of tick parasitism are particularly susceptible to the effect of basophil mediators other than histamine.     

Ixodes holocyclus: Kinetics of cutaneous basophil responses in naive,and actively and passively sensitized guinea pigs

In guinea pigs, macroscopic cutaneous reactions to initial (primary) Ixodes holocyclus feeding were first apparent at 96 hr post-tick attachment, peaked at 7 days (5 mm), and were gone by Day 14. Microscopic analyses of these primary tick feeding sites at 12, 24, 48, 72, and 96 hr post-attachment revealed the dominance of mononuclear cells (63–94% of the infiltrate) at all times. Neutrophil levels were high initially (34% of the infiltrate), but quickly subsided to 6–15% of the cellular response. Eosinophils were essentially absent from primary sites, comprising only 1–3% of the infiltrate. Basophils were absent initially, but accumulated in small but significant numbers (12% of the infiltrate) at the epidermal-dermal border by 96 hr post-tick attachment, 3 days prior to maximum erythematous skin reactions. In sensitized and challenged (secondary) animals, erythematous reactions in response to secondary tick feeding were apparent as early as 18 hr post-tick attachment with peak responses at 48 hr (3–4 mm). Cutaneous leukocyte responses to challenge feedings were quantitated at 12, 24, 48, and 72 hr posttick attachment and consisted initially (12–24 hr) of a strong mononuclear cell response (72–79% of the infiltrate) that was replaced by a dominant basophil response at 48 and 72 hr. Strong cutaneous basophil responses coincided with a significant level of tick rejection. Eosinophils and neutrophils were virtually absent from these secondary responses. Other ixodid tick-guinea pig systems have a relatively stronger eosinophil response, and both basophils and eosinophils participate in immune resistance in these systems. This suggests that basophils alone mediate resistance in the I. holocyclus system. Recipients of immune serum or immune cells expressed significant cutaneous basophil responses 72 hr post-tick challenge (23 and 39% of the infiltrate, respectively). Eosinophil and neutrophil responses were weak (1–6%), whereas mononuclear cells were dominant (54–72%). Recipients of immune lymph node cells expressed a cutaneous basophilia approximately two-thirds (69%) of that observed in actively sensitized and challenged hosts, whereas immune serum recipients recruited less than one-third (31%) as many basophils. In summary, these results indicate that T-cell- and antibody-dependent mechanisms recruit infiltrates of basophils that are responsible for acquired immune resistance to I. holocyclus in guinea pigs.     

The role of endogenous porphyrins in laser therapy of experimental skin wounds

The role of endogenous porphyrins in the effect of laser irradiation on the superoxide dismutase (SOD) activity of wound exudate and rat leukocyte activity has been studied on models of aseptic incised skin wounds. Wounds were irradiated with a He-Ne laser (632.8 nm, 1.5 J/cm²) on the 2nd, 3rd, and 4th days after the beginning of the experiment. Irradiation effects were evaluated by the SOD activity (NBT test) and the activity of leukocytes of the wound exudate (as a chemiluminescent response to opsonized zymosan). It was found that in animals subjected to laser irradiation, the SOD activity sharply increased. This effect depended on endogenous porphyrin concentration and was retained throughout the experiment. The SOD activity in unirradiated animals decreased from the 2nd to the 5th day of experiment. The evaluation of the activity of wound exudate leukocytes did not reveal any distinct dependence of the effect on the concentration of endogenous porphyrins.     

A Chitosan Based,Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the ''gold-standard'' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.¹ Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.² Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin ''solders'' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.³ Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.⁴In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed ''SurgiLux''. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.⁵ Without this ''activation'' process, SurgiLux films are readily removed.^6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.^6-10     

Transplantation of the future coronal suture on the dura mater of 3- to 4-month-old rats.

From fetal rat skulls, tissue containing the presumptive coronal suture was excised and transplanted onto the exposed dura mater of adult rats. Donor animals were sacrificed at specific ages determined by developmental stages of the suture as occurs in the rat. Thus, sacrifices were made at (a) the 19th day of fetal life when a blastema formation in the suture is not yet observable; (b) the 20th day when blastema formation becomes apparent, and (c) the 21st day when the blastema is clearly visible. From the results of these experiments, the following conclusions could be drawn: (1) transplants from 19- and 20-day-old fetuses are not capable of autonomous development in host tissue; continuation and regulation of this process occur in situ only; (2) within the connective tissue of the 19- and 20-day transplants the process of suture formation is arrested; instead chondrogenetic activity occurs, resulting in the production of ectopic cartilage, and (3) when formation of the blastema has been completed prior to transplantation, i.e. at the 21st day, its capability of producing a suture in the host tissue remains unimpaired. Sutures originating from these transplants do not ossify, not even when left in situ for 2 or 3 weeks. These findings suggest the existence of an osteogenesis-inhibiting mechanism located in embryonic sutural tissue, being transmitted to the developing dura.     

Effect of photosensitizers pheophorbide a and protoporphyrin IX on skin wound healing upon low-intensity laser irradiation

The effect of photosensitizer with subsequent He-Ne (632.8 nm; 3 mW/cm²) laser irradiation on experimental skin wound healing has been studied. Pheophorbide a and protoporphyrin IX were used as photosensitizers. It was found that application of the photosensitizer and subsequent laser irradiation, first, decreased the amount and the functional activity of leukocytes in the wound exudate and, second, inhibited the SOD activity as compared with that of the control group. Moreover, pheophorbide and protoporphyrin practically did not affect the total healing period but decreased the length of the inflammation stage. It was supposed that these effects are related to generation of reactive oxygen species during irradiation.     

Glucose and mannitol diffusion in human dura mater

An in vitro experimental study of the control of the human dura mater optical properties at administration of aqueous solutions of glucose and mannitol has been presented. The significant increase of the dura mater optical transmittance under action of immersion liquids has been demonstrated. Diffusion coefficients of glucose and mannitol in the human dura mater tissue at 20 degrees C have been estimated as (1.63 +/- 0.29) x 10(-6)cm(2)/s and as (1.31 +/- 0.41) x 10(-6) cm(2)/s, respectively. Experiments show that administration of immersion liquids allows for the effective control of tissue optical characteristics that make dura mater more transparent, thereby increasing the ability of light penetration through the tissue.     

Numerical investigation of thermal response of laser-irradiated biological tissue phantoms embedded with gold nanoshells

The work presented in this paper focuses on numerically investigating the thermal response of gold nanoshells-embedded biological tissue phantoms with potential applications into photo-thermal therapy wherein the interest is in destroying the cancerous cells with minimum damage to the surrounding healthy cells. The tissue phantom has been irradiated with a pico-second laser. Radiative transfer equation (RTE) has been employed to model the light-tissue interaction using discrete ordinate method (DOM). For determining the temperature distribution inside the tissue phantom, the RTE has been solved in combination with a generalized non-Fourier heat conduction model namely the dual phase lag bio-heat transfer model. The numerical code comprising the coupled RTE-bio-heat transfer equation, developed as a part of the current work, has been benchmarked against the experimental as well as the numerical results available in the literature. It has been demonstrated that the temperature of the optical inhomogeneity inside the biological tissue phantom embedded with gold nanoshells is relatively higher than that of the baseline case (no nanoshells) for the same laser power and operation time. The study clearly underlines the impact of nanoshell concentration and its size on the thermal response of the biological tissue sample. The comparative study concerned with the size and concentration of nanoshells showed that 60 nm nanoshells with concentration of 5×10¹⁵ mm⁻³ result into the temperature levels that are optimum for the irreversible destruction of cancer infected cells in the context of photo-thermal therapy. To the best of the knowledge of the authors, the present study is one of the first attempts to quantify the influence of gold nanoshells on the temperature distributions inside the biological tissue phantoms upon laser irradiation using the dual phase lag heat conduction model.     

Effect of ionizing radiation on the properties of rat liver phosphoprotein phosphatase

Phosphoproteidphosphatase (3.1.3.16) of high specificity for lysil-tRNA-synthetase (6.1.1.6) and proteins of high-molecular-weight multienzyme complex of aminoacyl-tRNA-synthetases (6.1.1.) was isolated from rat liver. Irradiation of animals with an absolutely lethal dose of 0.21 C/kg decreased phosphoproteidphosphatase activity: a 3-4-fold decrease was noted 1 hr following irradiation. The activity of the enzyme isolated 24 hr after irradiation increased but did not reach the control level.     

Systemic expression of cutaneous basophil hypersensitivity.

Guinea pigs primed for cutaneous basophil hypersensitivity (CBH) with several soluble proteins or with sheep erythrocytes developed a systemic, delayed-onset, maculopapular rash when challenged parenterally with specific antigen. The rash was most readily induced 5 to 7 days after immunization, at a time when local CBH skin test reactivity was also optimal. Miscroscopically, the rash resembled local CBH skin test reactions, being comprised of a papillary dermal infiltrate of basophils and lymphocytes and a striking dilatation and compaction of superficial venules. In addition to the systemic rash, animals expressing systemic CBH (SCBH) exhibited a striking eosinophilia at 24 hr which gave way to basophilia at 48 hr. Focal collections of eosinophils, and of smaller numbers of basophils, were found in the lungs and spleen; both eosinophils and basophils infiltrated the medulla of the thymus. Thus, basophil-rich infiltrations are favored in the skin even after systemic challenge with antigen and occur only to a much smaller extent in other organs where eosinophils may predominate. These differences in the response of various organs to challenge with parenteral antigen suggest that as yet unidentified local factors play a determinative role in regulating the inflammatory response. The pathogenesis of SCBH is not yet established, but it shares many of the properties of local CBH: histology, carrier specificity, development early after sensitization in the absence of detectable antibodies. Passive transfer has not been accomplished with serum alone but has been achieved irregularly with cells plus serum. SCBH may serve as a useful model for several disease states in man characterized by a systemic rash and eosinophilia, including certain types of drug reaction.     

IgG1 antibody-dependent mediator release after passive systemic sensitization of basophils arriving at cutaneous basophil hypersensitivity reactions

When antigen is injected into a 24-hr cutaneous basophil hypersensitivity (CBH) reaction of an actively sensitized guinea pig, local basophils degranulate and release histamine. This reaction is called cutaneous basophil anaphylaxis and may be antibody mediated. We now report passive sensitization of basophils at CBH sites by systemic transfer of anti-picryl immune serum. Keyhole limpet hemocyanin- (KLH) immunized animals were skin tested with KLH to elicit 24-hr CBH reactions at day 7. Anti-picryl serum was injected i.v. at various times. On day 7, blue dye was injected i.v., and then 24-hr CBH sites vs nearby normal skin were challenged with 0.1 microgram picryl-human serum albumin (Pic-HSA). An immediate increase in vascular permeability (blueing) was noted at normal skin sites due to systemic passive cutaneous anaphylaxis (PCA), and augmented blueing occurred at CBH sites compared with normal skin. Systemic passive sensitization of CBH sites occurred when antiserum was administered as little as 1 hr before challenge of CBH site. However, local administration of anti-picryl serum (as in a local PCA reaction) was not able to sensitize tissue basophils, whether antigen was administered locally or systemically. The serum factor that mediated cutaneous basophil anaphylaxis was heat-stable (56 degrees C X 4 hr) 7S IgG1 antibody. Electron microscopy of Pic-HSA-challenged CBH sites in animals that received IgG1 antibody showed that local basophils undergo anaphylactic degranulation by exocytosis. These studies suggest that basophils arriving at CBH reactions are sensitized for anaphylactic function by antibody that can be acquired in the circulation, but possibly not at the local site.     

Effect of total profound hypothermia on the relation between cells in the skin of the white rat at different ages

In experiments performed on 190 white inbred rats of various age, effects of total deep hypothermia on mitotic activity of basal epidermocytes, quantity of non-pigmented granular dendrocytes and lymphocytes of epidermis, tissue dermal basophils has been studied. Age difference in changes of the parameters examined at cold effect have been stated. A suggestion is made on existence in the skin of a local regularity system "basal epidermocyte--non-pigmented granular dendrocyte--intraepidermal lymphocyte--tissue dermal basophils", participating in regulation of protective properties of the organ.