Differences in growth and vascularization of ectopic menstrual and non-menstrual endometrial tissue in mouse models of endometriosis.

STUDY QUESTION: Is there a difference in the growth and vascularization between murine endometriotic lesions originating from menstrual or non-menstrual endometrial fragments? SUMMARY ANSWER: Endometriotic lesions developing from menstrual and non-menstrual tissue fragments share many similarities, but also exhibit distinct differences in growth and vascularization, particularly under exogenous estrogen stimulation. WHAT IS KNOWN ALREADY: Mouse models are increasingly used in endometriosis research. For this purpose, menstrual or non-menstrual endometrial fragments serve for the induction of endometriotic lesions. So far, these two fragment types have never been directly compared under identical experimental conditions. STUDY DESIGN, SIZE, DURATION: This was a prospective experimental study in a murine peritoneal and dorsal skinfold chamber model of endometriosis. Endometrial tissue fragments from menstruated (n = 15) and non-menstruated (n = 21) C57BL/6 mice were simultaneously transplanted into the peritoneal cavity or dorsal skinfold chamber of non-ovariectomized (non-ovx, n = 17), ovariectomized (ovx, n = 17) and ovariectomized, estrogen-substituted (ovx+E2, n = 17) recipient animals and analyzed throughout an observation period of 28 and 14 days, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: The engraftment, growth and vascularization of the newly developing endometriotic lesions were analyzed by means of high-resolution ultrasound imaging, intravital fluorescence microscopy, histology and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Menstrual and non-menstrual tissue fragments developed into peritoneal endometriotic lesions without differences in growth, microvessel density and cell proliferation in non-ovx mice. Lesion formation out of both fragment types was markedly suppressed in ovx mice. In case of non-menstrual tissue fragments, this effect could be reversed by estrogen supplementation. In contrast, endometriotic lesions originating from menstrual tissue fragments exhibited a significantly smaller volume in ovx+E2 mice, which may be due to a reduced hormone sensitivity. Moreover, menstrual tissue fragments showed a delayed vascularization and a reduced blood perfusion after transplantation into dorsal skinfold chambers when compared to non-menstrual tissue fragments, indicating different vascularization modes of the two fragment types. To limit the role of chance, the experiments were conducted under standardized laboratory conditions. Statistical significance was accepted for a value of P < 0.05. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Endometriotic lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The present study significantly contributes to the characterization of common murine endometriosis models. These models represent important tools for studies focusing on the basic mechanisms of endometriosis and the development of novel therapeutic strategies for the treatment of this frequent gynecological disease. The presented findings indicate that the combination of different experimental models and approaches may be the most appropriate strategy to study the pathophysiology and drug sensitivity of a complex disease such as endometriosis under preclinical conditions. STUDY FUNDING/COMPETING INTEREST(S): There was no specific funding of this study. The authors have no conflicts of interest to declare.

Angiogenic effects of mesenchymal stem cells in combination with different scaffold materials.

Tissue survival in regenerative tissue engineering requires rapid vascularization, which is influenced by scaffold material and seeded cell selection. Poly-l-lactide-co-glycolide (PLGA) and beta-tricalcium phosphate (ß-TCP) are well-established biomaterials with angiogenic effects because of their material properties. Given the importance of the seeded cell type as a co-factor for vascularization, mesenchymal stem cells (MSCs) are known to have high angiogenic potential. We hypothesized that PLGA and ß-TCP scaffolds seeded with MSCs would effectively induce a potent angiogenic response. Therefore, we studied the angiogenic effects after implanting PLGA and ß-TCP scaffolds seeded with isogeneic MSCs in vivo. Fifty-six BALB/c mice were equally divided into seven groups and underwent implantation of the dorsal skinfold chambers. Two MSC groups were seeded on collagen-coated PLGA or ß-TCP scaffolds, whereas groups 3-6 received collagen-coated or uncoated scaffolds without MSCs. No scaffold implantation was performed for group 7, which served as the control. Angiogenesis was assessed in vivo via intravital fluorescence microscopy. Angiogenic responses were noted on all scaffolds, whereupon MSC angiogenic response was significantly enhanced on days 6 and 10. Additionally, a comparison of biomaterials indicated increased angiogenic activity for ß-TCP scaffolds compared with PLGA scaffolds. In conclusion, seeding ß-TCP scaffolds with MSCs can accelerate vitalization and a combination of both significantly improves angiogenesis.

Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte-endothelial cell interaction.

Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte-endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte-endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs.

Effects of a new piezoelectric device on periosteal microcirculation after subperiosteal preparation.

INTRODUCTION: Subperiosteal preparation using a periosteal elevator leads to disturbances of local periosteal microcirculation. Soft-tissue damage can usually be considerably reduced using piezoelectric technology. For this reason, we investigated the effects of a novel piezoelectric device on local periosteal microcirculation and compared this approach with the conventional preparation of the periosteum using a periosteal elevator. MATERIAL AND METHODS: A total of 20 Lewis rats were randomly assigned to one of two groups. Subperiosteal preparation was performed using either a piezoelectric device or a periosteal elevator. Intravital microscopy was performed immediately after the procedure as well as three and eight days postoperatively. Statistical analysis of microcirculatory parameters was performed offline using analysis of variance (ANOVA) on ranks (p<0.05). RESULTS: At all time points investigated, intravital microscopy demonstrated significantly higher levels of periosteal perfusion in the group of rats that underwent piezosurgery than in the group of rats that underwent treatment with a periosteal elevator. CONCLUSION: The use of a piezoelectric device for subperiosteal preparation is associated with better periosteal microcirculation than the use of a conventional periosteal elevator. As a result, piezoelectric devices can be expected to have a positive effect on bone metabolism.

Alendronate reduces periosteal microperfusion in vivo.

Objectives: Bisphosphonates are known to induce a severe adverse effect known as medication-related osteonecrosis of the jaw (MRONJ). Previous studies have proven the impact of bisphosphonates on microperfusion; therefore, this study aimed to investigate alendronate-induced microcirculatory reactions in the calvarial periosteum of rats. Study design: Bone chambers were implanted into 48 Lewis rats. Microhemodynamics, inflammatory parameters, functional capillary density and defect healing were examined after alendronate treatment for two and six weeks using repetitive intravital fluorescence microscopy for two weeks. Results: Microhemodynamics remained unchanged. In alendronate-treated rats, inflammation was slightly increased, functional capillary density was significantly reduced (day 10: controls 100.45 ± 5.38 cm/cm2, two weeks alendronate treatment 44.77 ± 3.55 cm/cm2, six weeks alendronate treatment 27.54 ± 2.23 cm/cm2) and defect healing was decelerated. The changes in functional capillary density and defect healing were dose-dependent. Conclusion: The bisphosphonate alendronate has a significant negative impact on periosteal microperfusion in vivo. This could be a promising target for the treatment of MRONJ.

Caloric Restriction: A Novel Conditioning Strategy to Improve the Survival of Ischemically Challenged Musculocutaneous Random Pattern Flaps.

Caloric restriction (CR) is a cost-effective and easy-to-perform approach to counteracting surgical stress. The present study therefore evaluates the tissue-protective effects of a 30% CR in musculocutaneous flaps undergoing ischemia. For this purpose, a well-established murine dorsal skinfold chamber model, in combination with random pattern musculocutaneous flaps, was used. C57BL/6N mice were divided at random into a CR group (n = 8) and a control group with unrestricted access to standard chow (n = 8). The CR animals were subjected to a 30% reduction in caloric intake for 10 days before flap elevation. Intravital fluorescence microscopy was carried out on days 1, 3, 5, 7 and 10 after flap elevation to assess the nutritive blood perfusion, angiogenesis and flap necrosis. Subsequently, the flap tissue was harvested for additional histological and immunohistochemical analyses. The CR-treated animals exhibited a significantly higher functional capillary density and more newly formed microvessels within the flap tissue when compared to the controls; this was associated with a significantly higher flap survival rate. Immunohistochemical analyses showed a decreased invasion of myeloperoxidase-positive neutrophilic granulocytes into the flap tissue of the CR-treated mice. Moreover, the detection of cleaved caspase-3 revealed fewer cells undergoing apoptosis in the transition zone between the vital and necrotic tissue in the flaps of the CR-treated mice. These results demonstrate that a CR of 30% effectively prevents flap necrosis by maintaining microperfusion on a capillary level and inhibiting inflammation under ischemic stress. Hence, CR represents a promising novel conditioning strategy for improving the survival of musculocutaneous flaps with random pattern perfusion.

Bromelain Protects Critically Perfused Musculocutaneous Flap Tissue from Necrosis.

Bromelain has previously been shown to prevent ischemia-induced necrosis in different types of tissues. In the present study, we, therefore, evaluated for the first time, the tissue-protective effects of bromelain in musculocutaneous flaps in mice. Adult C57BL/6N mice were randomly assigned to a bromelain treatment group and a control group. The animals were treated daily with intraperitoneal injections of 20 mg/kg bromelain or saline (control), starting 1 h before the flap elevation throughout a 10-day observation period. The random-pattern musculocutaneous flaps were raised on the backs of the animals and mounted into a dorsal skinfold chamber. Angiogenesis, nutritive blood perfusion and flap necrosis were quantitatively analyzed by means of repeated intravital fluorescence microscopy over 10 days after surgery. After the last microscopy, the flaps were harvested for additional histological and immunohistochemical analyses. Bromelain reduced necrosis of the critically perfused flap tissue by ~25%. The bromelain-treated flaps also exhibited a significantly higher functional microvessel density and an elevated formation of newly developed microvessels in the transition zone between the vital and necrotic tissues when compared to the controls. Immunohistochemical analyses demonstrated a markedly lower invasion of the myeloperoxidase-positive neutrophilic granulocytes and a significantly reduced number of cleaved caspase 3-positive apoptotic cells in the transition zone of bromelain-treated musculocutaneous flaps. These findings indicate that bromelain prevents flap necrosis by maintaining nutritive tissue perfusion and by suppressing ischemia-induced inflammation and apoptosis. Hence, bromelain may represent a promising compound to prevent ischemia-induced flap necrosis in clinical practice.

Boosting Tissue Vascularization: Nanofat as a Potential Source of Functional Microvessel Segments.

Nanofat is increasingly applied in plastic surgery for the improvement of scar quality and skin rejuvenation. However, little is known about the underlying regenerative mechanisms. Therefore, we herein investigated nanofat grafts in a murine dorsal skinfold chamber model. Nanofat generated from subcutaneous, inguinal adipose tissue of green fluorescent protein (GFP)+ C57BL/6 male and female donor mice was injected intracutaneously into dorsal skinfold chambers of gender-matched GFP- wild-type mice. The vascularization and tissue composition of the grafted nanofat were analyzed by means of intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. The freshly generated nanofat consisted of small fragments of perilipin+ adipocytes surrounded by Sirius red+ collagen fibers and still contained intact CD31+/GFP+ vessel segments. After transplantation into the dorsal skinfold chamber, these vessel segments survived and developed interconnections to the surrounding CD31+/GFP- host microvasculature. Accordingly, the grafted nanofat rapidly vascularized and formed new microvascular networks with a high functional microvessel density on day 14 without marked differences between male and female mice. Even though further research is needed to confirm these findings, the present study suggests that nanofat boosts tissue vascularization. Thus, nanofat may represent a versatile resource for many applications in tissue engineering and regenerative medicine.

Real-Time Imaging of Immune Modulation by Cannabinoids Using Intravital Fluorescence Microscopy.

Introduction: The endocannabinoid system (ECS) is an endogenous regulatory system involved in a wide range of physiologic and disease processes. Study of ECS regulation provides novel drug targets for disease treatment. Intravital microscopy (IVM), a microscopy-based imaging method that allows the observation of cells and cell-cell interactions within various tissues and organs in vivo, has been utilized to study tissues and cells in their physiologic microenvironment. This article reviews the current state of the IVM techniques used in ECS-related inflammation research. Methodological Aspects of IVM: IVM with focus on conventional fluorescent microscope has been introduced in investigation of microcirculatory function and the behavior of individual circulating cells in an in vivo environment. Experimental setting, tissue protection under physiologic condition, and microscopical observation are described. Application of IVM in Experimental Inflammatory Disorders: Using IVM to investigate the effects of immune modulation by cannabinoids is extensively reviewed. The inflammatory disorders include sepsis, arthritis, diabetes, interstitial cystitis, and inflammatory conditions in the central nervous system and eyes. Conclusion: IVM is a critical tool in cannabinoid and immunology research. It has been applied to investigate the role of the ECS in physiologic and disease processes. This review demonstrates that the IVM technique provides a unique means in understanding ECS regulation on immune responses in diseases under their physical conditions, which could not be achieved by other methods.

Improved Vascularization and Survival of White Compared to Brown Adipose Tissue Grafts in the Dorsal Skinfold Chamber.

Fat grafting is a frequently applied procedure in plastic surgery for volume reconstruction. Moreover, the transplantation of white adipose tissue (WAT) and brown adipose tissue (BAT) increasingly gains interest in preclinical research for the treatment of obesity-related metabolic defects. Therefore, we herein directly compared the vascularization capacity and survival of WAT and BAT grafts. For this purpose, size-matched grafts isolated from the inguinal WAT pad and the interscapular BAT depot of C57BL/6N donor mice were syngeneically transplanted into the dorsal skinfold chamber of recipient animals. The vascularization and survival of the grafts were analyzed by means of intravital fluorescence microscopy, histology, and immunohistochemistry over an observation period of 14 days. WAT grafts showed an identical microvascular architecture and functional microvessel density as native WAT. In contrast, BAT grafts developed an erratic microvasculature with a significantly lower functional microvessel density when compared to native BAT. Accordingly, they also contained a markedly lower number of CD31-positive microvessels, which was associated with a massive loss of perilipin-positive adipocytes. These findings indicate that in contrast to WAT grafts, BAT grafts exhibit an impaired vascularization capacity and survival, which may be due to their higher metabolic demand. Hence, future studies should focus on the establishment of strategies to improve the engraftment of transplanted BAT.

Blood-Brain Barrier (BBB) Permeability and Transport Measurement In Vitro and In Vivo.

Quantification of the blood-brain barrier (BBB) permeability and transport in brain tissue is crucial in understanding brain disorders and developing systemic and non-systemic drug delivery strategies to the brain. This chapter summarizes BBB permeability measurement in vitro (Part I) and the in vivo non-invasive methods for quantifying the BBB permeability to solutes and brain tissue transport in rat brain by employing intravital multiphoton microscopy and a curving fitting method by using an unsteady mass transfer mathematical model (Part II).

In Vivo Assessment of Thermosensitive Liposomes for the Treatment of Port Wine Stains by Antifibrinolytic Site-Specific Pharmaco-Laser Therapy.

Antifibrinolytic site-specific pharmaco-laser therapy (SSPLT) is an experimental treatment modality for refractory port wine stains (PWS). Conceptually, antifibrinolytic drugs encapsulated in thermosensitive liposomes are delivered to thrombi that form in semi-photocoagulated PWS blood vessels after conventional laser treatment. Local release of antifibrinolytics is induced by mild hyperthermia, resulting in hyperthrombosis and complete occlusion of the target blood vessel (clinical endpoint). In this study, 20 thermosensitive liposomal formulations containing tranexamic acid (TA) were assayed for physicochemical properties, TA:lipid ratio, encapsulation efficiency, and endovesicular TA concentration. Two candidate formulations (DPPC:DSPE-PEG, DPPC:MPPC:DSPE-PEG) were selected based on optimal properties and analyzed for heat-induced TA release at body temperature (T), phase transition temperature (Tm), and at T > Tm. The effect of plasma on liposomal stability at 37 °C was determined, and the association of liposomes with platelets was examined by flow cytometry. The accumulation of PEGylated phosphocholine liposomes in laser-induced thrombi was investigated in a hamster dorsal skinfold model and intravital fluorescence microscopy. Both formulations did not release TA at 37 °C. Near-complete TA release was achieved at Tm within 2.0-2.5 min of heating, which was accelerated at T > Tm. Plasma exerted a stabilizing effect on both formulations. Liposomes showed mild association with platelets. Despite positive in vitro results, fluorescently labeled liposomes did not sufficiently accumulate in laser-induced thrombi in hamsters to warrant their use in antifibrinolytic SSPLT, which can be solved by coupling thrombus-targeting ligands to the liposomes.

University of Wisconsin solution for the xeno-free storage of adipose tissue-derived microvascular fragments.

Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen-glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

Oxytocin effects on experimental skin wound healing.

OBJECTIVE: Oxytocin (OXY) has significant effects on mammalian behavior. Next to its role in lactation and social interactions, it is described to support better wound healing as well. However, direct OXY effects on wound healing and the regeneration of the microvascular network are still not clarified. We therefore examined the effects of OXY and an OXY receptor antagonist [atosiban (ATO)] on skin wound healing, focusing on epithelialization and neovascularization. METHODS: Skin wound healing has been assessed using intravital fluorescence microscopy in a model of full dermal thickness wounds in the dorsal skin fold chamber of hairless mice. Animals received repetitive low or high doses of OXY or ATO. Morphological and cellular characterization of skin tissue repair was performed by histology and in vitro cell assays. RESULTS: The assessment of skin tissue repair using this therapy regimen showed that OXY and ATO had no major influence on epithelialization, neovascularization, wound cellularity, or inflammation. Moreover, OXY and ATO did neither stimulate nor deteriorate keratinocyte or fibroblast migration and proliferation. CONCLUSION: In summary, this study is the first to demonstrate that OXY application does not impair skin wound healing or cell behavior. However, until now, the used transmitter system seems not to be clarified in detail, and it might be proposed that it is associated with the stress response of the organism to various stimuli.

Dorsal skinfold chamber models in mice.

Background/purpose: The use of dorsal skinfold chamber models has substantially improved the understanding of micro-vascularisation in pathophysiology over the last eight decades. It allows in vivo pathophysiological studies of vascularisation over a continuous period of time. The dorsal skinfold chamber is an attractive technique for monitoring the vascularisation of autologous or allogenic transplants, wound healing, tumorigenesis and compatibility of biomaterial implants. To further reduce the animals' discomfort while carrying the dorsal skinfold chamber, we developed a smaller chamber (the Leipzig Dorsal Skinfold Chamber) and summarized the commercial available chamber models. In addition we compared our model to the common chamber. Methods: The Leipzig Dorsal Skinfold Chamber was applied to 66 C57Bl/6 female mice with a mean weight of 22 g. Angiogenesis within the dorsal skinfold chamber was evaluated after injection of fluorescein isothiocyanate dextran with an Axio Scope microscope. The mean vessel density within the dorsal skinfold chamber was assessed over a period of 21 days at five different time points. The gained data were compared to previous results using a bigger and heavier dorsal skinfold model in mice. A PubMed and a patent search were performed and all papers related to "dorsal skinfold chamber" from 1st of January 2006 to 31st of December 2015 were evaluated regarding the dorsal skinfold chamber models and their technical improvements. The main models are described and compared to our titanium Leipzig Dorsal Skinfold Chamber model. Results: The Leipzig Dorsal Skinfold Chamber fulfils all requirements of continuous in vivo models known from previous chamber models while reducing irritation to the mice. Five different chamber models have been identified showing substantial regional diversity. The newly elaborated titanium dorsal skinfold chamber may replace the pre-existing titanium chamber model used in Germany so far, as it is smaller and lighter than the former ones. However, the new chamber does not reach the advantages of already existing chamber models used in Asia and the US, which are smaller and lighter. Conclusion: Elaborating a smaller and lighter dorsal skinfold chamber allows research studies on smaller animals and reduces the animals' discomfort while carrying the chamber. Greater research exchange should be done to spread the use of smaller and lighter chamber models.

Measuring Vascular Permeability In Vivo.

Over the past decades, in vivo vascular permeability measurements have provided significant insight into vascular functions in physiological and pathophysiological conditions such as the response to pro- and anti-angiogenic signaling, abnormality of tumor vasculature and its normalization, and delivery and efficacy of therapeutic agents. Different approaches for vascular permeability measurements have been established. Here, we describe and discuss a conventional 2D imaging method to measure vascular permeability, which was originally documented by Gerlowski and Jain in 1986 (Microvasc Res 31:288-305, 1986) and further developed by Yuan et al. in the early 1990s (Microvasc Res 45:269-289, 1993; Cancer Res 54:352-3356, 1994), and our recently developed 3D imaging method, which advances the approach originally described by Brown et al. in 2001 (Nat Med 7:864-868, 2001).

Effects of volume resuscitation on the microcirculation in animal models of lipopolysaccharide sepsis: a systematic review.

BACKGROUND: Recent research has identified an increased rate of mortality associated with fluid bolus therapy for severe sepsis and septic shock, but the mechanisms are still not well understood. Fluid resuscitation therapy administered for sepsis and septic shock targets restoration of the macro-circulation, but the pathogenesis of sepsis is complex and includes microcirculatory dysfunction. OBJECTIVE: The objective of the study is to systematically review data comparing the effects of different types of fluid resuscitation on the microcirculation in clinically relevant animal models of lipopolysaccharide-induced sepsis. METHODS: A structured search of PubMed/MEDLINE and EMBASE for relevant publications from 1 January 1990 to 31 December 2015 was performed, in accordance with PRISMA guidelines. RESULTS: The number of published papers on sepsis and the microcirculation has increased steadily over the last 25 years. We identified 11 experimental animal studies comparing the effects of different fluid resuscitation regimens on the microcirculation. Heterogeneity precluded any meta-analysis. CONCLUSIONS: Few animal model studies have been published comparing the microcirculatory effects of different types of fluid resuscitation for sepsis and septic shock. Biologically relevant animal model studies remain necessary to enhance understanding regarding the mechanisms by which fluid resuscitation affects the microcirculation and to facilitate the transfer of basic science discoveries to clinical applications.

Mechanical Stabilization of Mouse Carotid Artery for In Vivo Intravital Microscopy Imaging of Atherogenesis.

We present here a procedure that allows real-time high-resolution multichannel imaging of early atherosclerotic lesions of live mice, by dramatically reducing the respiratory and pulsatile movements of the athero-susceptible carotid artery, without significantly altering blood flow dynamics. This surgical preparation can be combined with the use of various fluorescent probes and reporter mice to simultaneously visualize the dynamics of inflammatory leukocytes, platelets, or even subcellular structures. Stabilization of the tissue renders it suitable for two-photon laser scanning microscopic imaging and allows tracking the behavior of inflammatory cells in three dimensions.

IV and IP administration of rhodamine in visualization of WBC-BBB interactions in cerebral vessels.

Epi-illuminescence intravital fluorescence microscopy has been employed to study leukocyte-endothelial interactions in a number of brain pathologies. Historically, dyes such as Rhodamine 6G have been injected intravenously. However, intravenous injections can predispose experimental animals to a multitude of complications and requires a high degree of technical skill. Here, we study the efficacy of injecting Rhodamine 6G into the peritoneum (IP) for the purpose of analyzing leukocyte-endothelial interactions through a cranial window during real time intravital microscopy. After examining the number of rolling and adherent leukocytes through a cranial window, we found no advantage to the intravenous injection (IV). Additionally, we tested blood from both routes of injection by flow cytometry to gain a very precise picture of the two methods. The two routes of administration failed to show any difference in the ability to detect cells. The study supports the notion that IP Rhodamine 6G works as efficaciously as IV and should be considered a viable alternative in experimental design for investigations employing intravital microscopy. Facilitated intravital studies will allow for more exploration into cerebral pathologies and allow for more rapid translation from the laboratory to the patient with less chance of experimental error from failed IV access.