1. Volume 13, Issue 1, Pages 229-236 (January 2006)
Rapid Intravascular Injection into Limb Skeletal Muscle: A Damage Assessment Study 
Hechmi Toumi, Julia Hegge, Vladimir Subbotin, Mark Noble, Hans Herweijer, Thomas M. Best, James E. Hagstrom 
Molecular Therapy 
Volume 13, Issue 1, Pages (January 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
H&E-stained cross sections of rat gastrocnemius muscle at various times after rapid intravascular injection of saline. (A) Low-power view of gastrocnemius muscle (100× original magnification) 1 h after injection into great saphenous vein (3 ml/18 s). (B) High-power view of gastrocnemius muscle (400× original magnification) 1 h after injection into great saphenous vein (3 ml/18 s). (C) Cross section of muscle (100× original magnification) 1 h after injection into iliac artery (10 ml/18 s). (D) Cross section of muscle (100× original magnification) 24 h after injection into great saphenous vein (3 ml/18 s). (E) Cross section of muscle (200× original magnification) 24 h after injection into iliac artery (10 ml/18 s). (F) Cross section of muscle (400× original magnification) 24 h after bupivicaine injection. Note the interstitial edema at 1 h postinjection (A–C) and its resolution by 24 h (D and E). Female Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN, USA) weighing 120 to 140 g were used for all studies. All procedures were carried out in accordance with the NRC Guide for the Care and Use of Laboratory Animals and were approved by the Mirus Bio IACUC. Animals were anesthetized with isoflurane throughout each procedure and given analgesics for the first 48 h after each procedure. The intra-arterial (iliac artery) injections were performed as previously described [4]. For intravenous injections, rats were anesthetized with isoflurane (1–2%) and prepped for surgery. A latex band was wrapped tightly around the upper right hind limb and held securely in place with a hemostat. A skin incision was made to expose the great saphenous vein extending from the ankle to just below the knee [6]. A 25-gauge needle catheter was inserted into the distal saphenous vein and advanced about 10 mm. The catheter tubing was connected to a two-way adapter that connected to two syringe pumps (Harvard Apparatus, Holliston, MA, USA) for delivery of papaverine and the DNA delivery solution. The vasodilator papaverine (0.25 mg) was delivered in a total volume of 1.5 ml saline at a rate of 10 ml/min. Five minutes after papaverine, 3.0 ml of saline was infused at a rate of 10 ml/min. Two minutes after the injection, the catheter was removed and the tourniquet was released. Bleeding was controlled with a cotton swab, and the skin was closed with sutures.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Evans Blue (EB) staining of gastrocnemius muscles following intravascular injection. All muscles were harvested 20 hours post-EB staining (A) EB staining of uninjected naive rat (negative control; 400× original magnification). (B) EB staining of bupivicaine-treated muscle (positive control for damage; 400× original magnification). (C) EB staining of limb muscle from venous-injected rat (no stained myofibers detected in vast majority of cross sections; 400× original magnification). (D) EB staining of one of the two localized areas displaying myofiber membrane damage following intravenous injection (i.e., myofibers positive for EB staining; 200× original magnification). (E) Percentage of EB-positive cells observed in cross section of the gastrocnemius muscle group following intra-artery or intravenous or bupivicaine injection. The number of EB-positive cells in relation to the total number of cells is indicated in parentheses. For bupivicaine injections rats were anesthetized as described and a 27-gauge needle was percutaneously inserted into the gastrocnemius muscle starting distally and advancing along the length of the muscle until the needle was fully inserted [9]. The needle was then slowly withdrawn while injecting 0.5 ml of a 0.375% bupivicaine solution (Abbott Laboratories). EB (Sigma; E2129) was prepared as a 1% solution dissolved in phosphate-buffered saline and filtered through a 0.2-μm syringe filter. Four hours after an intravascular injection procedure (intra-arterial or intravenous), rats were anesthetized with isoflurane (1–2%) and injected intraperitoneally with EB (1% by volume of total body mass) using a 3-cc syringe and a 27-gauge needle [16]. Animals were euthanized and the gastrocnemius muscle group was harvested at 20 h after the EB injection and frozen in isopentane cooled by liquid nitrogen. The gastrocnemius muscle group referred to in this study includes five major muscles from the posterior position of the lower leg. These include the gastrocnemius, soleus, flexor digitorum longus, flexor digitorum superficialis, and tibialis caudalis. Muscles samples for immunohistochemistry or EB staining were stored at −80°C until further processing. For the EB staining, slides were viewed with a green-wavelength filter (Zeiss; excitation 546–580, emission 590). Images were saved using standardized exposure settings. For quantification, the numbers of EB-positive cells and total cells in each section were determined by manually counting cells in a cross area starting from the top edge of the section to the bottom edge and from the left edge to the right edge.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Accumulation of leukocytes into gastrocnemius or hamstring muscle groups at various time points after rapid arterial (iliac) or venous (great saphenous) injection. Injections were performed using saline alone (saline) or saline plus a non-expressing plasmid DNA construct. The plasmid DNA used in this study (pMIR51) consists of a pUC19 vector backbone with a muscle creatine kinase promoter/enhancer but contains no expressible mammalian sequences within the multiple cloning site. Immunohistochemical staining for ED1+ and ED2+ cells (A and B) 24 h after intra-arterial injection, (C and D) 24 h after intravenous injection, and (E and F) 24 h after bupivicaine injection (positive control) is shown at 400× original magnification. Arrows indicate ED1- or ED2-positive cells (i.e., red cells). (G–I) Quantitation of leukocyte accumulation in muscle following intra-arterial or intravenous injections. (G) CD43+ neutrophils; (H) ED1+ blood macrophages; (I) ED2+ tissue macrophages. Increases in leukocyte infiltration were statistically significant (P < 0.05) compared to naïve controls. For immunohistochemistry analysis transverse frozen sections (10 μm) of the gastrocnemius and hamstring muscles were cut on a cryostat (HM 505 N; Leica), allowed to air dry, and then fixed with 4% neutral-buffered formalin and washed in PBS. Sections were then incubated with the mouse anti-rat antibodies anti-ED1+, anti-ED2+, and anti-CD43 all diluted 1:200 (Serotec, Oxford, UK). Anti-ED1+ recognizes a single-chain glycoprotein predominantly expressed on membranes of blood macrophages, whereas anti-ED2+ reacts with the membrane antigen present on tissue resident macrophages. Anti-CD43 recognizes a glycoprotein on neutrophils, monocytes, and T lymphocytes. Sections were incubated for 30 min with the antibodies, washed in PBS, and then incubated with a Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:1000 for 30 min. After incubation, sections were washed in PBS and counterstained with Alexa 488 phalloidin (Molecular Probes; diluted 1:400) for actin and To-Pro3 (Molecular Probes; diluted 1:70,000) for nuclei. Each section was then covered with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and a glass coverslip. Evaluation of the stained sections was done using a Zeiss LSM 510 confocal microscope. Five random images (207 × 225 × 10 μm) from each section were selected without any knowledge of the presence of positive cells by viewing only in the green (actin) channel. Each selected image was scanned with standardized settings and then saved. The number of fluorescently labelled cells in each image was then counted manually, and the mean was used for statistical analysis. Means and standard deviations were calculated for each control and time point. A one-way ANOVA was used to compare differences between the groups. When significant treatment effects occurred, Fischer post hoc tests were used. In each case, the level of significance was established at P < 0.05. All analyses were carried out in a blinded fashion to the treatment condition.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 3
Accumulation of leukocytes into gastrocnemius or hamstring muscle groups at various time points after rapid arterial (iliac) or venous (great saphenous) injection. Injections were performed using saline alone (saline) or saline plus a non-expressing plasmid DNA construct. The plasmid DNA used in this study (pMIR51) consists of a pUC19 vector backbone with a muscle creatine kinase promoter/enhancer but contains no expressible mammalian sequences within the multiple cloning site. Immunohistochemical staining for ED1+ and ED2+ cells (A and B) 24 h after intra-arterial injection, (C and D) 24 h after intravenous injection, and (E and F) 24 h after bupivicaine injection (positive control) is shown at 400× original magnification. Arrows indicate ED1- or ED2-positive cells (i.e., red cells). (G–I) Quantitation of leukocyte accumulation in muscle following intra-arterial or intravenous injections. (G) CD43+ neutrophils; (H) ED1+ blood macrophages; (I) ED2+ tissue macrophages. Increases in leukocyte infiltration were statistically significant (P < 0.05) compared to naïve controls. For immunohistochemistry analysis transverse frozen sections (10 μm) of the gastrocnemius and hamstring muscles were cut on a cryostat (HM 505 N; Leica), allowed to air dry, and then fixed with 4% neutral-buffered formalin and washed in PBS. Sections were then incubated with the mouse anti-rat antibodies anti-ED1+, anti-ED2+, and anti-CD43 all diluted 1:200 (Serotec, Oxford, UK). Anti-ED1+ recognizes a single-chain glycoprotein predominantly expressed on membranes of blood macrophages, whereas anti-ED2+ reacts with the membrane antigen present on tissue resident macrophages. Anti-CD43 recognizes a glycoprotein on neutrophils, monocytes, and T lymphocytes. Sections were incubated for 30 min with the antibodies, washed in PBS, and then incubated with a Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:1000 for 30 min. After incubation, sections were washed in PBS and counterstained with Alexa 488 phalloidin (Molecular Probes; diluted 1:400) for actin and To-Pro3 (Molecular Probes; diluted 1:70,000) for nuclei. Each section was then covered with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and a glass coverslip. Evaluation of the stained sections was done using a Zeiss LSM 510 confocal microscope. Five random images (207 × 225 × 10 μm) from each section were selected without any knowledge of the presence of positive cells by viewing only in the green (actin) channel. Each selected image was scanned with standardized settings and then saved. The number of fluorescently labelled cells in each image was then counted manually, and the mean was used for statistical analysis. Means and standard deviations were calculated for each control and time point. A one-way ANOVA was used to compare differences between the groups. When significant treatment effects occurred, Fischer post hoc tests were used. In each case, the level of significance was established at P < 0.05. All analyses were carried out in a blinded fashion to the treatment condition.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions