穿支皮瓣Choke区动、静脉血流阻力对皮瓣存活的影响

目的　通过在同一组皮瓣中对choke血管的动、静脉阻力进行比较研究,探究血流跨过choke血管的动、静脉阻力大小关系及其对皮瓣存活的影响。 方法　将34只SD大鼠随机分成两组,A组保留右侧肋间后动脉+左侧肋间后静脉,B组保留右侧肋间后动脉+右侧髂腰静脉。测量保留的动、静脉之间的距离。术后7天,统计皮瓣存活率以及血管造影观察皮瓣区微血管形态;choke区取材,H&E染色,计算平均微血管密度;尾静脉采血,检测乳酸含量。 结果　A组保留的动、静脉之间的距离比B组短[(2.5±0.3) cm vs (3.7±0.2) cm,t=14.608,P<0.05）]。术后7 d,A组皮瓣存活率为100%,B组1号血管体区存活率为（67.0±13.1）%,4号血管体区的为100%,整块皮瓣存活率为（88.0±6.8）%;A组比B组血管增生明显[（24.0±3.9）vs (17.6±4.3),t=2.727,P=0.021];A组与B组乳酸均接近术前水平,差异无统计学意义[（8.0±0.8）mmol/L vs (8.4±0.4) mmol/L vs (7.5±0.6) mmol/L,P>0.05]。 结论　①血流跨过choke血管的静脉阻力大于动脉阻力;②缩短跨choke区的动、静脉血流单向距离有利于减小跨choke区所受的静脉阻力,提高皮瓣的存活率。     

大鼠背部穿支体区间choke血管变化规律初探

目的　设计可对大鼠背部穿支体区间choke血管进行直视观察的模型，并进一步探索choke血管扩增的规律。 方法　制作皮肤血管观察窗套件，选择左、右髂腰动脉穿支体区间的微血管吻合（即choke血管）作为目标观察区，并分时间点在体视显微镜下观察choke血管的扩增，测量并计算微血管在各时间点的“扩增率”，总结其扩增规律。 结果　设计的皮肤观察窗套件可清楚观察到大鼠皮肤血管的扩增过程，该观察窗套件还可根据观察对象的大小调整尺寸进而观察不同动物的choke血管。通过对大鼠背部穿支体区间choke血管观察发现第1h、1d、2d、3d、4d、6d、10d、16d目标观察区choke血管的“扩增率”分别是（1.00±0.00）、（1.11±0.08）、（1.25±0.17）、（1.36±0.22）、（1.85±0.33）、（1.82±0.38）、（1.54±0.39）和（1.83±0.45）。 结论　对于皮肤微血管的活体动物研究，皮窗结合体视显微镜观察是方便、有效的直视观察手段。大鼠两髂腰动脉穿支体间的choke血管在第4~6天时处于扩增高峰，逐渐减弱后在第10~16天出现二次扩增。     

L-精氨酸通过L-Arg-NO途径促进大鼠背部跨区皮瓣的成活

文题释义：跨区皮瓣：穿支血管体区是某一源动脉呈三维立体网状结构所供应的所有解剖区域,是设计皮瓣的解剖学基础。临床上大面积皮肤软组织缺损时常需扩大切取皮瓣,皮瓣内包含2个以上穿支血管体区,称为跨区皮瓣。Choke区：穿支血管呈树状分支,且口径逐渐减小,并与周围临近穿支血管形成血管网状连接,及“choke vessels”,两相邻穿支血管之间的区域称为Choke区。某一穿支血管不能通过choke区无限制向皮瓣远端供血,穿支皮瓣的缺血、坏死经常出现在choke区域,choke vessels成了穿支皮瓣血运的最大阻力。背景：有研究表明一氧化氮可有效改善皮瓣术后血供,促进皮瓣成活,但具体机制尚不清楚。 目的：通过实验验证L-Arg-NO途径在L-精氨酸促进大鼠背部跨区皮瓣成活中的作用。 方法：成功建立大鼠背部三穿支体跨区皮瓣模型的雄性SD大鼠81只,随机被分为3组,分别于术后即刻、术后1-7 d腹腔注射不同药物,L-Arg组注射L-精氨酸400 mg/(kg·d);L-NAME组注射一氧化氮合酶抑制剂40 mg/(kg·d);空白组注射等体积等渗氯化钠溶液。术后7 d观察皮瓣成活情况,计算皮瓣成活率;颈总动脉灌注明胶-氧化铅行血管造影,观察血管走形和分布;硝酸还原酶法测定术后即刻、1,3,5,7 d chokeⅡ区一氧化氮水平;苏木精-伊红染色观察choke Ⅱ区新生血管密度和管径;蛋白印记法检测术后3,7 d chokeⅡ区血管内皮生长因子和基质金属蛋白酶2蛋白表达。实验方案经解放军联勤保障部队第九四〇医院动物实验伦理委员会批准(批准号为2015KYLL046)。 结果与结论：①术后7 d,L-Arg组大鼠皮瓣成活率最高(89.47±3.17)%,3组比较差异有显著性意义(F=49.908,P < 0.001);②术后7 d,L-Arg组choke Ⅱ区血管结构较完整,走形清楚,扩张达到真性吻合的血管沿着皮瓣纵轴方向一直延伸到皮瓣末端,空白组和L-NAME组chokeⅡ区血管结构和走形杂乱,L-NAME组皮瓣末端血管结构消失;③L-Arg组一氧化氮水平术后开始升高,术后3 d达到高峰,之后逐渐下降;④术后7 d,L-Arg组新生血管密度和管径最高,3组比较差异有显著性意义(均P < 0.001);⑤术后3 d,L-Arg组血管内皮生长因子和基质金属蛋白酶2蛋白表达量最高,3组比较差异有显著性意义(均P < 0.05);术后7 d,L-Arg组血管内皮生长因子和基质金属蛋白酶2蛋白表达量最高,3组之间比较血管内皮生长因子差异有显著性意义(P < 0.05),基质金属蛋白酶2蛋白差异无显著性意义;⑥结果说明,一氧化氮可有效改善大鼠背部三穿支体皮瓣chokeⅡ区血供,促进chokeⅡ区微血管扩张和增生,而L-精氨酸可通过L-Arg-NO途径提高皮瓣术后组织内一氧化氮水平,促进皮瓣术后成活。ORCID: 0000-0002-1812-9457(李文波);0000-0003-2403-1082(高秋明) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

蒂部动脉位置对跨区穿支皮瓣成活的影响

目的 通过改变蒂部动脉的位置，探讨其对跨区穿支皮瓣成活的影响。 方法 将32只SD大鼠随机分成两组：对照组，保留右侧髂腰动、静脉；实验组，保留右侧髂腰静脉+左侧髂腰动脉。术后7 d，统计皮瓣成活率，血管造影观察choke动脉扩张情况，HE染色显微镜下测量choke动脉的腔内直径。 结果 术后7 d，实验组皮瓣成活率高于对照组，（85.5±2.9）% vs（68.9±2.6）%，P<0.001；两组choke1动脉扩张程度相当，（46.93±6.21）μm vs（46.79±6.69）μm，P>0.05，实验组choke2动脉扩张较对照组更明显，（66.08±6.78）μm vs（46.00±6.90）μm，P<0.001。 结论 蒂部动脉位置改变，可促进choke动脉扩张和皮瓣远端供血，从而提高跨区穿支皮瓣成活率。     

皮蒂在穿支筋膜蒂皮瓣中的作用探讨

目的　进一步阐明穿支筋膜皮瓣的皮蒂对皮瓣的静脉引流和动脉输入的作用。 方法　40只重约300g的雄性SD大鼠,在背部切取大小为9 cm×3 cm的皮瓣后,等分入以下4组: 穿支完好组(蒂部穿支保留完好)、动脉缺失组(结扎蒂部动脉)、静脉缺失组（结扎蒂部静脉）及穿支缺失组（结扎动静脉）。术后7 d使用激光多普勒血流仪监测皮瓣血液灌注,计算皮瓣坏死率,测量皮蒂血管的直径。 结果　激光多普勒血流仪提示穿支完好组与静脉缺失组的灌注模式相似,穿支缺失组与动脉缺失组的灌注模式相似。穿支完好组和静脉缺失组的血流灌注量更大。穿支缺失组皮瓣的坏死率（26±1）%与动脉缺失组皮瓣的坏死率（29±1）%无显著差异,但均显著高于穿支完好组（11±3）%和静脉缺失组（12±4）%皮瓣的坏死率（P<0.001）。静脉缺失组和穿支缺失组的皮肤基底部静脉网明显扩张,而动脉缺失组和穿支完好组的动脉网管径轻度增大。 结论　穿支筋膜皮瓣的皮蒂作为静脉引流额外通道的作用比其作为动脉输入的作用更加重要。     

跨区皮瓣切取后choke血管管径的定量分析及MMP-2与MMP-9的表达

目的　探讨MMP-2与MMP-9在Choke血管演变过程中的表达情况。 方法　SD 大鼠40 只,体重(310±10)g。在大鼠背部一侧切取髂腰动脉穿支为蒂,宽3 cm,长10 cm的皮瓣。术后将皮瓣分如5个时间点,在每个时间点切取髂腰动脉与肋间后动脉穿支之间的Choke区组织,利用Western blot、明胶酶谱法及免疫组化对MMP-2与MMP-9的表达情况进行分析。利用皮窗技术对Choke血管管径的变化进行定量分析。 结果　 Western blot结果检测表明皮瓣术后MMP-2、MMP-9的表达量均有所增高,MMP-9以术后1 d增高最为显著;明胶酶谱结果显示,术后MMP-9的活性明显增高,以术后1 d增高最为显著,而MMP-2的活性没有显著差异。MMP-2免疫阳性细胞主要表达在血管内皮,MMP-9主要表达在血管腔内及血管内皮。PGP9.5免疫组织化学显色,皮瓣切取后3d皮瓣内的轴突已经完全退变。 结论　通过对皮瓣内血管演变过程进行定量分析,在形态学上验证了跨区皮瓣术后前3 d静脉血“迷宫式回流”,之后主要通过轴型静脉进行回流的假说。在跨区皮瓣术后血管的形态学演变中MMP-9可能扮演比MMP-2更重要的角色。     

静脉超引流与动脉增压对皮瓣存活的影响

目的比较静脉超引流与动脉增压对穿支皮瓣微循环血流动力学的影响,为降低皮瓣的坏死率,提高其存活面积提供一定的依据。方法雄性SD大鼠60只,体重450~550g,分成实验组A、实验组B及对照组,每组20只。实验组A,分离并结扎肋间后动脉保留伴行静脉;实验组B,分离并结扎肋间后静脉保留伴行动脉;对照组,肋间后动、静脉均结扎切断。在术后6h、1d、2d、3d、7d,超声多普勒测量两个"choke区"的经皮血流量和氧分压,7d后测量皮瓣存活面积和进行明胶氧化铅血管造影。结果术后7d,实验组A皮瓣几乎全部存活(98.2±1.6)%,实验组B皮瓣存活面积(74.78±5.91)%,对照组存活面积(60.3±7.8)%(两两比较P0.01)。从术后6h到7d,实验组A相比于实验组B和对照组血流量和氧分压是最高的(均P0.05)。实验组A,肋间后静脉和髂腰穿支吻合区"choke vessel 1"区血管吻合丰富,而且肋间后穿支和胸背穿支吻合区"choke vessel 2"区比实验组B和对照组吻合都要明显;实验组B出现了动静脉的吻合,对照组皮瓣远端严重坏死,"choke vessel 2"区血管吻合不明显。结论静脉超引流比动脉增压效果更明显,更能有效地改善微循环血流灌注效果,提高皮瓣的存活率。     

外科延迟术对跨区皮瓣多血管体间微循环重构的影响

目的建立小鼠背部单穿支和多穿支跨区皮瓣模型,探讨3种延迟术对皮瓣微循环重构的影响,术后中央choke区内皮型一氧化氮合酶(e-NOS)、基质金属蛋白酶-2(MMP-2)的变化以及该区域血流对其形态学的影响。方法 Balb/c小鼠100只,随机分为对照组(10只)及实验A、B、C组(每组30只)。A组结扎右胸背穿支;B组结扎2条右侧穿支;C组仅保留左胸背穿支。各实验组分别于术前、术后6h、1d、3d、5d、7d用激光多普勒血流仪连续监测choke区,观察血管形态学变化,并取材进行HE染色,对目的蛋白的表达进行检测(Western blotting)及定位(免疫组织化学染色)。结果与术前相比,术后各组choke区血流增加明显,差异且有统计学意义(P0.01);此区血管曲度也明显增加,管径增粗,管壁增厚,蛋白含量增加,目的蛋白主要定位于血管内及其外周。结论术后各组choke区呈现不同的血流动力学与血管形态学变化特点,即微循环重构;以保留单穿支的延迟方式对跨区皮瓣choke区微循环重构影响最大;e-NOS、MMP-2参与了血管新生过程。     

联体皮瓣的静脉引流位置对动脉血流导向功能的影响

目的 探讨联体皮瓣的静脉引流位置对动脉血流导向功能的影响。方法 将72只SD大鼠随机分为4组:正常组不做任何处理;对照组仅保留右侧肋间后动、静脉;实验组1仅保留右侧肋间后动脉+左侧肋间后静脉;实验组2仅保留右侧肋间后动脉+左侧髂腰静脉。测量血管体之间距离。术后7 d,统计皮瓣存活率以及明胶氧化铅血管造影观察皮瓣区血管形态;choke 1区取材,HE染色,分析动脉管径扩大倍数;尾静脉采血,检测乳酸含量。结果 血管体Ⅰ离Ⅱ最近,离Ⅲ最远,差异有统计学意义（P<0.05）。术后7 d,对照组皮瓣存活率为（86.5±4.3）%,实验组1和实验组2皮瓣存活率均为100%;血管造影显示,在血管体Ⅲ与Ⅳ区域,对照组血管增生比较少,而实验组1比较明显,实验组2最明显;与正常组相比,实验组2动脉管径扩大最明显,其次为实验组1,差异有统计学意义（P<0.05）;对照组乳酸含量最高,差异有统计学意义（P<0.05）。结论 静脉引流位置的改变可造成动脉灌流与静脉引流效果的改变,进而改变皮瓣区域生理性血管网,提高存活率。     

激光多普勒对大鼠背部3种皮瓣模型的血流动力学特点研究

目的　利用激光多普勒对大鼠背部3种皮瓣的血流动力学特点进行研究。 方法　30只雄性SD大鼠,按照皮瓣切取方式等分为3组：穿支皮瓣组、穿支筋膜蒂皮瓣组及随意皮瓣组。采用激光多普勒血流仪对以上3组皮瓣的Ⅰ、Ⅱ与Ⅲ区在多个时间点进行测量。术后7 d,对皮瓣外观及髂腰动脉与骶尾部穿支血管间的筋膜血管网进行拍照。对3组大鼠的坏死率,及穿支皮瓣与穿支筋膜蒂皮瓣Ⅰ、Ⅱ及Ⅲ区术后即刻、术后1 d的血流灌注值进行统计学比较。 结果　穿支皮瓣、穿支筋膜蒂皮瓣及随意皮瓣的坏死率分别为0.087±0.07、0.071±0.05及0.267±0.11。3组皮瓣坏死率有极显著统计学差异,但穿支皮瓣与穿支筋膜蒂皮瓣的坏死率差异无统计学意义。术后即刻穿支皮瓣Ⅰ区与Ⅱ区的血流灌注值显著大于穿支筋膜蒂皮瓣的Ⅰ区与Ⅱ区;随意皮瓣3个血管区的血流灌注值在术后7 d内均小于穿支皮瓣与穿支筋膜蒂皮瓣。术后7 d,随意皮瓣骶尾部穿支与髂腰动脉穿支间的筋膜血管网管径显著增大。 结论　穿支皮瓣与穿支筋膜蒂皮瓣存活能力无显著差异。随意皮瓣存活主要依赖于蒂部与蒂部周围潜在穿支之间筋膜血管网的扩张。     

Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers

Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the static analysis, the heparan sulfate-containing nanofiber gels were found to persist in the tissue for up to 30 days and revealed excellent biocompatibility. Strikingly, as the nanofiber gel biodegraded, we observed the formation of a de novo vascularized connective tissue. In the dynamic experiments using the dorsal skinfold chamber, the material again demonstrated good biocompatibility, with minimal dilation of the microcirculation and only a few adherent leukocytes, monitored through intravital fluorescence microscopy. The new application of the dorsal skinfold model corroborated our findings from the traditional static histology, demonstrating the potential use of this technique to dynamically evaluate the biocompatibility of materials. The observed biocompatibility and development of new vascularized tissue using both techniques demonstrates the potential of these angiogenesis-promoting materials for a host of regenerative strategies.     

Quantitation and physiological characterization of angiogenic vessels in mice: effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenvironment.

A prerequisite for the development of novel angiogenic and anti-angiogenic agents is the availability of routine in vivo assays that permit 1) repeated, long-term quantitation of angiogenesis and 2) physiological characterization of angiogenic vessels. We report here the development of such an assay in mice. Using this assay, we tested the hypothesis that the physiological properties of angiogenic vessels governed by the microenvironment and vessel origin rather than the initial angiogenic stimulus. Gels containing basic fibroblast growth factor (bFGF) or vascular endothelial growth (VEGF) were implanted in transparent windows in the dorsal skin or cranium of mice. Vessels could be continuously and non-invasively monitored and easily quantified for more than 5 weeks after gel implantation. Newly formed vessels were first visible on day 4 in the cranial window and day 10 in the dorsal skinfold chamber, respectively. The number of vessels was dependent on the dose of bFGF and VEGF. At 3000 ng/ml, bFGF- and VEGF-induced blood vessels had similar diameters, red blood cell velocities, and microvascular permeability to albumin. However, red blood cell velocities and microvascular permeability to albumin were higher in the cranial window than in the dorsal skinfold chamber. Leukocyte-endothelial interaction was nearly zero in both sites. Thus, newly grown microvessels resembled vessels of granulation and neoplastic tissue in many aspects. Their physiological properties were mainly determined by the microenvironment, whereas the initial angiogenic response was stimulated by growth factors.     

Influence of poly(D,L-lactic-co-glycolic acid) microsphere degradation on arteriolar remodeling in the mouse dorsal skinfold window chamber

Poly(D,L-lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is widely used for drug delivery. However, the degradation of PLGA alters the local microenvironment and may influence tissue structure and/or function. Here, we studied whether PLGA degradation affects the structure of the arteriolar microcirculation through arteriogenic expansion of maximum lumenal diameters and/or the formation of new smooth muscle-coated vessels. Single microspheres comprised of 50:50 PLGA (521 +/- 52.7 microm diameter), 50:50 PLGA with bovine serum albumin (BSA) (547 +/- 62.2 microm), 85:15 PLGA (474 +/- 52.6 microm), or 85:15 PLGA with BSA (469 +/- 57.2 microm) were implanted into mouse dorsal skinfold window chambers, and longitudinal arteriolar diameter measurements were made in the presence of a vasodilator (10(-4)M adenosine) over 7 days. At the end of the 7-day period, the length density of all smooth muscle-coated microvessels was also determined. Implantation of the window chamber alone elicited a 22% increase in maximum arteriolar diameter. However, the addition of 85:15 and 50:50 PLGA microspheres, bearing either BSA or no protein, elicited a significant enhancement of this arteriogenic response, with final maximum arteriolar diameters ranging from 36 to 46% more than their original size. Interestingly, the influence of PLGA degradation on microvascular structure was limited to lumenal arteriolar expansion, as we observed no significant differences in length density of smooth muscle-coated microvessels. We conclude that the degradation of PLGA microspheres may elicit an arteriogenic response in subcutaneous tissue in the dorsal skinfold window chamber; however, it has no apparent effect on the total length of smooth muscle-coated microvasculature.     

In vivo biocompatibility and vascularization of biodegradable porous polyurethane scaffolds for tissue engineering

Scaffolds for tissue engineering should be biocompatible and stimulate rapid blood vessel ingrowth. Herein, we analyzed in vivo the biocompatibility and vascularization of three novel types of biodegradable porous polyurethane scaffolds. The polyurethane scaffolds, i.e., PU-S, PU-M and PU-F, were implanted into dorsal skinfold chambers of BALB/c mice. Using intravital fluorescence microscopy we analyzed vascularization of the implants and venular leukocyte–endothelial cell interaction in the surrounding host tissue over a 14 day period. Incorporation of the scaffolds was analyzed by histology, and a WST-1 assay was performed to evaluate their cell biocompatibility in vitro. Our results indicate that none of the polyurethane scaffolds was cytotoxic. Accordingly, rolling and adherent leukocytes in venules of the dorsal skinfold chamber were found in a physiological range after scaffold implantation and did not significantly differ between the groups, indicating a good in vivo biocompatibility. However, the three scaffolds induced a weak angiogenic response with a microvessel density of only ～47–60 and ～3–10 cm/cm² in the border and centre zones of the scaffolds at day 14 after implantation. Histology demonstrated that the scaffolds were incorporated in a granulation tissue, which exhibited only a few blood vessels and inflammatory cells. In conclusion, PU-S, PU-M and PU-F scaffolds may be used to generate tissue constructs which do not induce a strong inflammatory reaction after implantation into patients. However, the scaffolds should be further modified or conditioned in order to accelerate and improve the process of vascularization.     

Neovascularization of poly(ether ester) block-copolymer scaffolds in vivo: long-term investigations using intravital fluorescent microscopy

Poly(ether ester) block-copolymer scaffolds of different pore size were implanted into the dorsal skinfold chamber of balb/c mice. Using intravital fluorescent microscopy, the temporal course of neovascularization into these scaffolds was quantitatively analyzed. Three scaffold groups (diameter, 5 mm; 220-260 thickness, microm; n = 30) were implanted. Different pore sizes were evaluated: small (20-75 microm), medium (75-212 microm) and large pores (250-300 microm). Measurements were performed on days 8, 12, 16, and 20 in the surrounding normal tissue, in the border zone, and in the center of the scaffold. Standard microcirculatory parameters were assessed (plasma leakage, vessel diameter, red blood cell velocity, and functional vessel density). The large-pored scaffolds showed significantly higher functional vessel density in the border zone and in the center (days 8 and 12) compared with the scaffold with the small and medium-sized pores. These data correlated with a larger vessel diameter and a higher red blood cell velocity in the large-pored scaffold group. Interestingly, during the evaluation period the microcirculatory parameters on the edge of the scaffolds returned to values similar to those found in the surrounding tissue. In the center of the scaffold, however, neovascularization was still active 20 days after implantation. Plasma leakage and vessel diameter were higher in the center of the scaffold. Red blood cell velocity and functional vessel density were 50% lower than in the surrounding tissue. In conclusion, the dorsal skinfold chamber model in mice allows long-term study of blood vessel growth and remodeling in porous biomedical materials. The rate of vessel ingrowth into poly(ether ester) block-copolymer scaffolds is influenced by pore size and was highest in the scaffold with the largest pores. The data generated with this model contribute to knowledge about the development of functional vessels and tissue ingrowth into biomaterials.     

Biological effects of static magnetic fields on the microcirculatory blood flowin vivo: a preliminary report

There have been few studies of the effect of static magnetic fields on microcirculatory haemodynamics in vivo. The rat skinfold transparent chamber technique was used, which provides an excellent means of observing and quantifying direct in vivo microvascular haemodynamic responses to static magnetic fields up to 8 T. An intravital videomicroscope was used to measure the changes in blood flow before and after exposure to a magnetic field for 20 min in a horizontal type superconducting magnet with a bore 100 mm in diameter and 700 mm long. After exposure, microcirculatory blood flow showed an initial increase for about 5 min followed by a gradual decrease and a return to the control value. It is hypothesised that these changes represent rebound hyperaemia following reduced blood flow during exposure.     

Conduction properties of spinal cord axons in the myelin-deficient rat mutant.

Spinal cords of myelin-deficient and normal age-matched (control) rats were removed and their conduction and pharmacological properties studied in an in vitro brain slice chamber. The conduction velocity of the myelin-deficient dorsal column axons was reduced to about 25% of control axons; however, the amyelinated myelin-deficient axons displayed refractory periods and the ability to sustain high-frequency action potential discharge similar to that of dorsal column axons in control rats. Pharmacological results suggest that the myelin-deficient dorsal column axons qualitatively express a normal complement of ion channels and receptors. The demonstration of a normal representation of channels and receptors on these axons supports the proposal that the oligodendrocyte, and not the axon, is the site of the primary defect in the myelin-deficient rat mutant. It is concluded that, unlike acutely demyelinated axons which display marked frequency-dependent conduction block, amyelinated axons of the myelin-deficient rat spinal cord develop compensatory mechanisms to stabilize action potential conduction.     

In Vivo Imaging of Physiological Angiogenesis from Immature to Preovulatory Ovarian Follicles

To develop a model for the study of physiological angiogenesis, we transplanted ovarian follicles onto striated muscle tissue and analyzed the process of microvascularization in vivo using repeated fluorescence microscopy. Follicles were mechanically isolated from unstimulated as well as pregnant mare's serum gonadotropin (PMSG)- or PMSG/luteinizing hormone (LH)-stimulated Syrian golden hamster ovaries and were transplanted as free grafts into dorsal skinfold chambers of untreated or synchronized hamsters. Follicles lacking thecal cell layers did not vascularize regardless whether harvested from unstimulated or PMSG-stimulated animals, but underwent granulosa cell apoptosis, as indicated in vivo by nuclear condensation and fragmentation of bisbenzimide-stained follicular tissue. In contrast, all follicles at 48 hours after PMSG treatment with a multilayered thecal shell exhibited initial signs of angiogenesis within 3 days. Vascularization was completed within 7 to 10 days, comprising a dense glomerulum-like microvascular network. Nature and extent of vascularization of follicles harvested at 72 hours after either PMSG or PMSG/LH treatment did not notably differ from each other when transplanted into the respective synchronized animals. However, follicles with PMSG/LH treatment revealed significantly larger microvessel diameters and higher capillary blood perfusion compared to follicles with sole PMSG treatment, probably reflecting the adaptation to the increased functional demand upon the LH surge. Using the unique experimental approach of ovarian follicle transplantation in the dorsal skinfold chamber of Syrian golden hamsters, we could show in vivo the developmental stage-dependent vascularization of follicular grafts with sustained potential to meet their metabolic demand by increased blood perfusion.     

Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1)

Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2(-/-) (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.