Sham Injected Research Articles

180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (> 70%), DNA integrity (<8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P<0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P<0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.

Retinal Toxicity of Intravitreal Trastuzumab in a Rabbit Model: Preliminary Results of an Experimental Study

To evaluate the retinal toxicity of intravitreal trastuzumab in a rabbit model. Fourteen New Zealand albino rabbits, weighing between 2.5 and 3 kg were used for this study. In the first group (n=6), 0.1 mL trastuzumab 10 mg/mL was injected into the vitreous. In the second group (n=6), the same volume of sterile balanced saline solution was injected intravitreally (sham injection). Additionally, 0.1 mL of 2 other concentrations of trastuzumab (7.5 and 5 mg/mL, respectively), was injected into the vitreous of 2 rabbits. Slit-lamp and funduscopic examinations were performed and the animals were observed for 2 weeks for signs of inflammation, infection, and toxicity. An electroretinogram (ERG) was performed at baseline and 14 days after the injection. The animals were killed on day 14 and a histological examination was performed in the enucleated eyes. The clinical examination was unremarkable on either sham or intravitreal injection of trastuzumab. Conversely, the ERG was greatly affected and in 2 cases extinguished 14 days after trastuzumab injection. Consistent with electrophysiological abnormalities of the retina, signs of retinal edema in experimented eyes, suggesting morphologic retinal damage, were observed. In contrast, in the sham injected eyes, the ERG was normal without histopathologic retinal changes. Intravitreal trastuzumab seems to be toxic to the retina in albino rabbits even at a concentration of 5 mg/mL. Further studies are needed to evaluate the safety of intravitreal trastuzumab in models of choroidal neovascularization, as well as to obtain experience concerning the intravitreal toxicity of trastuzumab in primates too and not only in rabbits.

Effects of intrastriatal botulinum neurotoxin A on the behavior of Wistar rats

Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of dopaminergic and cholinergic circuits. In Parkinson's disease imbalanced cholinergic hyperactivity prevails in the striatum. As recently shown intrastiatal botulinum neurotoxin A (BoNT-A) improves motor function in hemiparkinsonian rats. Before going further steps in using intracerebral BoNT-injections as possible treatment we here explore whether pure BoNT-injections into normal rats’ striata affect their cognitive and emotional properties. Wistar rats were injected bilaterally with 1ng BoNT-A or vehicle (sham injection) into the striatum, whereas a naïve control group was left untreated. Locomotor activity, balance and coordination were assessed in open field and accelerod tests. Anxiety was evaluated in the open field and elevated plus maze. Spatial learning was assessed by radial and water maze tests. Intrastriatal BoNT-A, but also sham injections caused decreased motor activity and impaired balance and motor coordination of rats. Slight working memory deficits were observed in radial maze testing of both BoNT-A and sham injected animals arguing for a consequence of surgery rather than for a specific BoNT-A effect. In contrast, BoNT-A injected animals showed a reduced anxiety in open field and elevated plus maze compared to both sham-treated and naïve controls. As bilateral intrastriatal BoNT-A injections in normal rats do not cause cognitive impairments and reduce anxiety, and previous findings showed improvements of motor function in hemiparkinsonian rats following intrastriatal BoNT-A, it can be argued that intrastriatal BoNT-A could be a new therapeutic approach in Parkinson's disease.

Effect of neck injection and handler visibility on behavioral reactivity of beef steers1

The use of the neck region as an injection site in cattle is becoming routine. Use of a blind may reduce aversive behavior caused by the presence of the person administering the injection. To evaluate whether cattle react to the proximity of the stockperson or to the actual injection, one hundred twenty 10-mo-old Angus steers (298 +/- 28 kg of BW; mean +/- SD) were assigned to 1 of 4 treatment groups using a partial crossover design (neck/sham injection x blind/no blind) replicated over 2 d (3 d apart). Steers were restrained for a total of 60 s in a squeeze chute, with treatment being administered 20 s after entry. Animal reactivity was rated using 2 scoring methods, including a visual and an electronic score, for three 20-s intervals (pre-treatment, treatment, and posttreatment intervals). Flight speed (m/s) was used as a measure of aversion to the treatments and was taken upon release from the chute. No interactions (P > 0.10) were observed between the blind and injection treatments for any of the measurements taken. No treatment or day effect on flight speed (2.7 vs. 2.6 m/s; P > 0.03) was observed; however, the correlation between days (r = 0.74; P < 0.001) was significant. Visual scores indicated that injected steers were more agitated during the treatment interval than were the sham injected steers (1.9 vs. 1.6, respectively; P = 0.01). However, no differences (P > 0.10) were found between injection and sham injection treatments for any of the electronic scores. Steers exposed to the blind had lower electronic reactivity scores (P < 0.05) than those not exposed to the blind, which was in contrast to the results obtained for the visual scores (P < 0.05). Discrepancies between reactivity scores may be due to the difficulty of accurately assessing minor animal responses using the visual method. The presence of a handler during an injection procedure could be a contributor to the aversion response observed in cattle undergoing routine neck injections, and use of a blind helped to reduce the reactivity of the steers.

Granulocyte Colony-stimulating Factor Supports Liver Regeneration in a Small-for-size Liver Remnant Mouse Model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 μg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium-ionophore activation in the pig

In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitromatured porcine oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as handling controls. After subsequent in vitro-culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved with fresh in vitro-capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In Experiment 2, in vitro matured oocytes were injected with freshly ejaculated in vitro-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 μM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Caionophore treatment after injection of spermatozoa was ineffective at 100 μM, where at 50 μM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperminjected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed.

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, and 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.

Experimental immunization of rats with a Streptococcus mutans 59-kilodalton glucan-binding protein protects against dental caries

Glucan-binding proteins (GBPs) are theoretically important in the molecular pathogenesis of dental caries caused by Streptococcus mutans. The present study evaluated the ability of antibody induced by the S. mutans 59-kDa GBP (GBP59) to affect dental caries caused by experimental infection with S. mutans in a rodent model. Groups of 20-day-old rats were injected twice at 9-day intervals subcutaneously in the salivary gland vicinity with GBP59, glucosyltransferase (GTF), or phosphate-buffered saline (sham injection), each incorporated in an adjuvant. Two weeks after the second injection, GBP59- and GTF-injected rats contained significant levels of salivary immunoglobulin A and serum immunoglobulin G antibody to the respective injected antigens. However, cross-reacting antibody to S. mutans GTF or GBP59 was not induced by the respective antigen. Rats were then orally infected with S. mutans. After 71 days of infection, GBP59- and GTF-injected groups had smaller numbers of S. mutans on their molar surfaces, compared with the sham-injected infected group. Total, sulcal, and smooth-surface molar caries in the GBP59- and GTF-immunized S. mutans-infected groups were each significantly lower (P < or = 0.003) than the respective measures of caries in the sham injected infected group. The results of this investigation demonstrate that immunization with S. mutans GBP59 induces an immune response in rats that can interfere with the accumulation of S. mutans and can reduce the level of dental caries caused by this cariogenic streptococcus. Furthermore, the protective immunity induced by either GBP59 or GTF appears to result from antibodies to independent epitopes since these two S. mutans components do not have a close antigenic relationship.

Assessment of acute pulmonary edema in dogs by electrical impedance imaging.

Acute pulmonary edema was assessed quantitatively in 12 experiments on six anesthetized dogs. Thirty-two copper foil electrodes were placed around each animal's thorax at the level of the third intercostal space. A real-time electrical impedance tomograph was used to form images of the electrical admittivity of the thorax in and near the plane of these electrodes. The lungs were identified by studying the change in admittivity associated with inspiration. Mean admittivity in these lung regions was assessed at 40-min intervals for the next 3-6 hours. In six control experiments, each having a duration of 200 min, the initial admittivity of the lung regions was 102 +/- 16(SD) mS/m. Lung admittivity usually increased during the first 80 min, and then remained within 2 mS/m of its value at 80 min for the remaining 120 min. In six experiments with pulmonary edema, an initial period of change followed by stability was observed. When stability had been attained, 0.07 ml/kg of oleic acid was injected slowly into a central venous site. Five animals received oleic acid, the sixth received a sham injection of saline. In the five receiving oleic acid, lung admittivity rose steadily for the remainder of the experiment. The increase in lung admittivity in these five animals was between 4 and 16 mS/m. In the sham injected experiment, the lung admittivity changed by 1 mS/m. We conclude that impedance imaging can provide semiquantitative assessment of the development of acute pulmonary edema.

Neither intraocular grafts of retinal cell homogenates nor live non-retinal neurons produce behavioral recovery in rats with light-damaged retinas.

Previously we have observed that fetal retinal cells grafted to the subretinal space of blind rats produced a functional recovery as determined by testing the visual inhibition of the startle response. Following those studies, we performed experiments to test whether the injection itself, cell by-products, or unrelated neural cells could also produce an effect. Visual function was tested by examining the inhibitory effect of a brief light flash (300 lx) on the acoustic startle response to an immediately following intense noise burst in light blinded Fischer 344 rats. Animals were tested before and after grafts of fetal retinal cell homogenates, dissociated perinatal cerebellar cells, and sham injections in the subretinal space. Behavioral testing continued every 2 wk for 14 wk after the graft. In the pretests, the light flash inhibited the startle response, maximal at intervals of 40-70 ms with recovery thereafter. In contrast, after exposure for 4 wk to fluorescent light (300 lx) and a rest in a normal 12/12 h light/dark environment the rats showed reflex facilitation to the light, maximal at an interval of 110 ms, followed by a late period of reflex inhibition. The light flash had no effect on other rats that had been blinded by bilateral enucleation. Light blinded animals receiving either cerebellar grafts or retinal cell homogenates were no different in performance from their sham injected control animals. The present data suggest that neither subretinal injections of neural cells nor non-specific neurochemical factors are able to elicit a positive behavioral response in visually impaired animals.

Neither intraocular grafts of retinal cell homogenates nor live non-retinal neurons produce behavioral recovery in rats with light-damaged retinas

Previously we have observed that fetal retinal cells grafted to the subretinal space of blind rats produced a functional recovery as determined by testing the visual inhibition of the startle response. Following those studies, we performed experiments to test whether the injection itself, cell by-products, or unrelated neural cells could also produce an effect. Visual function was tested by examining the inhibitory effect of a brief light flash (3001x) on the acoustic startle response to an immediately following intense noise burst in light blinded Fischer 344 rats. Animals were tested before and after grafts of fetal retinal cell homogenates, dissociated perinatal cerebellar cells, and sham injections in the subretinal space. Behavioral testing continued every 2 wk for 14 wk after the graft. In the pretests, the light flash inhibited the startle response, maximal at intervals of 40–70 ms with recovery thereafter. In contrast, after exposure for 4 wk to fluorescent light (3001x) and a rest in a normal 12/12 h light/ dark environment the rats showed reflex facilitation to the light, maximal at an interval of 110 ms, followed by a late period of reflex inhibition. The light flash had no effect on other rats that had been blinded by bilateral enucleation. Light blinded animals receiving either cerebellar grafts or retinal cell homogenates were no different in performance from their sham injected control animals. The present data suggest that neither subretinal injections of neural cells nor non-specific neurochemical factors are able to elicit a positive behavioral response in visually impaired animals.

Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon.

The total Ca2+ content of the endoplasmic reticulum and the total Ca2+ and Mg2+ content of mitochondria were determined by electron probe microanalysis of rat liver rapidly frozen in vivo following brief (5-15 s) stimulation with vasopressin or prolonged (10-12 min) stimulation with vasopressin + glucagon. Brief vasopressin injection into the anterior mesenteric vein released 1.8 +/- 0.3 (S.D.) mmol of Ca2+/kg dry weight, from the rough endoplasmic reticulum (p less than 0.01), reducing Ca2+ content of the endoplasmic reticulum from 4.4 +/- 0.2 (S.E.) (controls) to 2.6 +/- 0.2 mmol of Ca2+/kg dry weight. Following vasopressin injection, endoplasmic reticulum Ca2+ was also significantly (p less than 0.025) lower than that in brief sham injected animals (3.5 +/- 0.2 mmol/kg dry weight). Mitochondrial Ca2+ was between 1.0 and 2.3 (+/-0.2) mmol/kg dry weight of mitochondrion, under all conditions studied, and no significant differences were observed. Both hormonal and brief sham injection into the anterior mesenteric vein increased mitochondrial Mg2+ from 42 (+/-0.8) to 49 (+/-1.8) mmol/kg dry weight (p less than 0.05). Hormonal stimulation of Mg2+ uptake was further confirmed by injection of vasopressin + glucagon into the jugular vein (to avoid any stimulation of the liver by the anterior mesenteric vein injection itself); mitochondrial Mg2+ increased from 43 (+/-0.9) (10-min sham) to 57 (+/-1.3) mmol/kg dry weight, with 10-min vasopressin + glucagon injection (p less than 0.01). These results demonstrate that hormones can release Ca2+ from the endoplasmic reticulum and modulate mitochondrial Mg2+ content in vivo without causing detectable changes in mitochondrial Ca2+.

Effect of estradiol on tissue glycogen metabolism in exercised oophorectomized rats.

The effect of both physiological and pharmacological doses of estradiol on exercise performance and tissue glycogen utilization was determined in oophorectomized estradiol-replaced (ER) rats. Doses of beta-estradiol 3-benzoate (0.02, 0.04, 0.1, 0.2, 1, 2, 4, or 10 micrograms.0.1 ml of sunflower oil-1.100 g body wt-1) were injected 5 days/wk for 4 wk. Controls were sham injected (SI). After treatment, the animals were run to exhaustion on a motorized treadmill. ER animals receiving the 0.02-microgram dose ran significantly longer and completed more total work than the SI group. ER animals receiving doses of greater than or equal to 0.04 microgram ran longer and performed more work than the 0.02-microgram group. At exhaustion, myocardial glycogen content was significantly decreased in animals that were ER with less than or equal to 0.1 microgram, whereas those replaced with doses greater than 0.1 microgram utilized significantly less glycogen. With the 10-micrograms dose no significant decrease in heart glycogen content was observed at exhaustion. A submaximal 2-h run significantly reduced glycogen content in heart, red and white portions of the vastus lateralis, and the livers of SI animals. The latter effect was attenuated in skeletal muscle and liver, and there was no effect in the hearts of the ER animals receiving 2 micrograms. These data indicate that estradiol replacement in oophorectomized rats influenced myocardial glycogen utilization during exhaustive exercise and spared tissue glycogen during submaximal exercise. These glycogen sparing effects may have contributed to the significant improvements in exercise performance observed in this study.