In the run up to the World Congress of Microcirculation, Beijing in September this year (20th-24th), we are looking at one of our more common applications in various perspectives: The pre-clinical use of the hind limb ischemia assessment techniques to develop novel therapies for peripheral arterial diseases and related applications.
The series includes Hind Limb Ischemia Techniques, Cell Treatments, Drug Therapies & Treatment Devices and Physiological studies.
Here, in the third episode, we briefly consider recent use of the hind limb ischemia model for the assessment of treatments with drugs and devices of peripheral arterial disease in studies on vascularisation following femoral artery occlusion.
We hope this provides useful background information on current uses of the measurement, practicalities. Ultimately we aim to help you make a well-informed choice on the equipment options available to you.
If you’d like to be kept informed regarding the release of the future articles in this series, please sign up to our newsletter at the bottom of the page. If you have any questions or comments on the above information or our applications, please don’t hesitate to contact us.
Drug therapies
A wide range of drugs and mechanisms have been shown to aid angiogenesis and post ischaemia tissue repair, with improved blood flow shown by moorLDI2 / moorFLPI-2 and the hind limb ischemia model:
Dong et al demonstrated the role of Interleukin-33 in facilitating blood flow restoration in the ischemic hindlimb.
Lewis et al increased the bioavailability of nitric oxide (a potent vasodilator), tested with NO-generating hydrogel (PA-YK-NO) or vehicle (CaCl2) injected into SS hindlimbs immediately following surgery. This failed to improve neovascularisation following hind limb ischemia in sickle cell disease mice and actually decreased perfusion (32% for vehicle, 19% for PA-YK-NO-treated mice (p<0.05).
Luo et al, performed a range of in vitro murine studies on sotagliflozin, an anti-hyperglycemia sodium-glucose cotransporter-2 (SGLT2) inhibitor, followed by in vivo assessments of hind limb blood flow with sotagliflozin (10 mg/kg) directly injected into gastrocnemius muscle of the left hindlimb once every 3 days for 3 weeks. This effectively promoted the formation of functional blood vessels, leading to significant recovery of blood perfusion in diabetic hind limb ischemia mice.
The Traditional Chinese medicines (1) Shuxuetong (SXT) and (2) Ruan jian qing mai (RJQM) were assessed by Sun et al and Zhu et al, respectively. 1, Following hind limb ischemia surgery, mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system in conjunction with a wide range of biochemical assays. 2, Dose dependence of was assessed in 3 RJQM groups compared with standard sodium beraprost (BPS) therapy: BPS (15.6 μg/kg) and RJQM (0.462, 0.924, and 1.848 g/kg) were administered daily for 14 consecutive days. LDI results showed no significant advantage for RJQM, at any dose, compared with BPS nor untreated hind limb ischemia. However, levels of inflammation, fibrosis and several biomarkers were improved in treatment groups.
Tong et al investigated the effect of 2% hydrogen gas inhalation on ischaemia-reperfusion injury (IR; 3 hours ischaemia, by elastic rubber band above the greater trochanter, and 4 hours reperfusion). No significant differences in LDI blood flow were found between sham, IR or IR+H2. However, muscle infarct area was reduced for IR+H2 compared with IR (P<0.05); endoplasmic reticulum stress, assessed by increase in GRP78 expression, was lower in the IR + H2 group than in the IR group (P<0.05).
Treatment Devices
Devices take many forms; large scale devices for the treatment of ischaemia include the opto-electronic, red light stimulation, Weihrauch et al, and extracorporeal shock wave therapy, used by Sung et al.
On a much smaller scale, the AngioTube, of Zohar et al, is a biodegradable engineered macro-vessel surrounded by a cylindrical micro-channel array designed to support physiological flow distribution and enable integration with living capillaries. In vivo function of the cell-seeded AngioTube, wrapped with cell-seeded gel, was demonstrated by direct anastomosis with the femoral artery in a rat hindlimb model; paw perfusion, assessed by moorFLPI-2 LSI, was significantly higher than control (p<0.01).
The overlap between a cell binding approach and a ‘device’ to enhance the cell environment reaches a smaller scale device with the development and use of PEG diacrylate by Sun et al: (PEGDA)/glycidyl methacrylate-HA (GMHA)-based cryogel system seeded with VEGF-transfected human tonsil-derived mesenchymal stem cells to induce enhanced neovascularization in murine hind-limb ischemia. Addition of GMHA allowed the PEGDA cryogel to enhance cell metabolism, including attachment and proliferation. The cell-seeded cryogel successfully facilitated regeneration of blood flow and regeneration of damaged tissue.
A nano scale device was developed by Zhou et al. Their study showed that EC-specific S1pr2 loss-of-function significantly enhanced post-ischemic angiogenesis and improved blood flow recovery upon femoral artery ligation, whereas the EC-specific S1pr2 gain-of-function severely hindered post-ischemic angiogenesis and reduced blood flow recovery in ischemic limbs. They developed RGD-peptide magnetic nanoparticles packaging S1pr2-siRNA which specifically targeted ECs and achieved an efficient silencing of S1pr2 expression in ECs in vivo. This EC-targeted strategy to dampen S1pr2 significantly enhanced post-ischemic angiogenesis and boosted blood perfusion (p<0.01 at day 14) after hind limb ischemia (see LDI images, above).
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References: Drug Therapies
Dong Q, Tian J, Zheng W, Fan Q, Wu X, Tang Y, Liu T, Yin H.
Interleukin-33 protects mice against hindlimb ischemic injury by enhancing endothelial angiogenesis.
Int Immunopharmacol. 2022 Aug;109:108850.
doi.org/10.1016/j.intimp.2022.108850
Lewis CV, Sellak H, Hansen L, Joseph G, Hurtado J, Archer DR, Jun H-W, Brown LA and Taylor WR.
Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice.
Laboratory Investigation volume 102, pages 805–813 (2022).
doi.org/10.1038/s41374-022-00780-0
Luo L-L, Han J-X, Wu S-R & Kasim V.
Intramuscular injection of sotagliflozin promotes neovascularization in diabetic mice through enhancing skeletal muscle cells paracrine function.
Acta Pharmacologica Sinica volume 43, pages 2636–2650 (2022).
doi.org/10.1038/s41401-022-00889-4
Sun W, Zhang L, Fang Z, Han L, Wang Q, Leng Y, Li M, Xue Y, Wu Y, Li Z, Wang H, Chen L.
Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.
J Ethnopharmacol. 2022 Jun 28;292:115166.
doi.org/10.1016/j.jep.2022.115166
Tong J, Zhang Y, Yu P, Liu J, Mei XL, Meng J.
Protective Effect of Hydrogen Gas on Mouse Hind Limb Ischemia-Reperfusion Injury.
J Surg Res. 2021 Oct;266:148-159.
doi.org/10.1016/j.jss.2021.03.046
Zhu D, Jia C, Cai T, Li J, Feng X, Chen N, Zhao C, Wang Y, Cao Y, and Cao Y.
Ruan Jian Qing Mai Recipe Inhibits the Inflammatory Response in Acute Lower Limb Ischemic Mice through the JAK2/STAT3 Pathway.
Evid Based Complement Alternat Med. 2022 Aug 18;2022:2481022.
doi.org/10.1155/2022/2481022
References: Treatment Devices
Sun W, Choi JH, Choi YH, Im SG, So K-H, Hwang NS.
VEGF-overexpressed Human Tonsil-derived Mesenchymal Stem Cells with PEG/HA-based Cryogels for Therapeutic Angiogenesis.
Biotechnology and Bioprocess Engineering volume 27, pages 17–29 (2022).
doi.org/10.1007/s12257-021-0061-x
Sung P-H, Yin T-C, Chai, H-T, Chiang JY, Chen C-H, Huang C-R, Yip H-K.
Extracorporeal ShockWave Therapy Salvages Critical Limb Ischemia in B6 Mice through Upregulating Cell Proliferation Signaling and Angiogenesis.
Biomedicines. 2022 Jan 6;10(1):117.
doi.org/10.3390/biomedicines10010117
Weihrauch D, Keszler A, Lindemer B, Krolikowski J, Lohr NL.
Red light stimulates vasodilation through extracellular vesicle trafficking.
J Photochem Photobiol B. 2021 Jul;220:112212.
doi.org/10.1016/j.jphotobiol.2021.112212
Zhou C, Kuang Y, Li Q, Duan Y, Liu X, Yue J, Chen X, Liu J, Zhang Y, Zhang L.
Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway.
Theranostics. 2022 Jul 4;12(11):5172-5188.
doi.org/10.7150/thno.71585
Zohar B, Debbi L, Machour M, Nachum N, Redenski I, Epshtein M, Korin N, Levenberg S.
A micro-channel array in a tissue engineered vessel graft guides vascular morphogenesis for anastomosis with self-assembled vascular networks.
Acta Biomater. 2023 Jun;163:182-193.
doi.org/10.1016/j.actbio.2022.05.026