For the experiments, P7 animals were decapitated, whereas P14, P21, P28, P42, P56, and P56–70 animals were anesthetized (100 mg/kg ketamine (CP-Pharma, Burgdorf, Germany), 10 mg/kg Xylazin (CP-Pharma)), and perfused with PBS/heparin (Ratiopharm, Ulm, Germany). Therefore, a constant pressure of 0.7 mL/min was generated with a peristaltic pump. After perfusion for 10–15 min, the animals were dissected. For all ages, the brains were removed from the skull and separated into hemispheres for cryosections, PCR, and western blot analysis.
Ketamine
Manufactured by CP-Pharma
Sourced in Germany
Ketamine is a general anesthetic used for the induction and maintenance of anesthesia. It is a white crystalline powder that is typically administered via injection. Ketamine is primarily used in veterinary medicine and for certain medical procedures in humans.
Automatically generated - may contain errors
Lab products found in correlation
Xylazin, by CP-Pharma (4 mentions)
Xylavet, by CP-Pharma (2 mentions)
Propofol, by CP-Pharma (2 mentions)
Dexmedetomidine, by CP-Pharma (2 mentions)
Levomethadone, by MSD (2 mentions)
Xylazine, by CP-Pharma (2 mentions)
Pbs heparin, by Ratiopharm (1 mentions)
Female lewis rats, by Charles River Laboratories (1 mentions)
Vicryl 3, by Johnson & Johnson (1 mentions)
Roti histokitt, by Carl Roth (1 mentions)
Frigomobil, by Reichert Technologies (1 mentions)
Xylazine, by Bayer (1 mentions)
Cepetor, by CP-Pharma (1 mentions)
Finepointe rc units, by Data Sciences International (1 mentions)
Flexivent, by SCIREQ (1 mentions)
Eg 1160 embedding center, by Leica camera (1 mentions)
Tp1020 semi enclosed benchtop tissue processor, by Leica camera (1 mentions)
Rm2155 microtome, by Leica camera (1 mentions)
16 protocols using ketamine
1
Perfusion and Brain Dissection Protocol
2
Astrocyte Maturation and LRP1 Function
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For the investigation of the influence of LRP1 on the maturation of astrocytes and their functionality, three developmental stages were analyzed (P21, P28, and P56-P70, summarized as adult). At the stage-specific age either control or knockout animals were anesthetized with 100 mg/kg ketamine (CP-Pharma, Burgdorf, Germany), 10 mg/kg Xylazin (CP-Pharma) in 0.9% NaCl. Afterward, the animals were transcardially perfused with phosphate buffered saline (PBS; 137 mM sodium chloride, 3 mM potassium chloride, 6.5 mM disodium hydrogen phosphate, 1.5 mM potassium dihydrogen phosphate; pH 7.3) and subsequently with 4% (w/v) paraformaldehyde (PFA) when the tissue was used for immunohistochemistry. Tissue for the RNA and protein isolation was prepared and transferred to a dish containing PBS. Then the hippocampus was removed and frozen in liquid nitrogen until further use.
3
Experimental Autoimmune Encephalomyelitis Induction in Lewis Rats
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Female Lewis rats with an age of 6–8 weeks were purchased from Charles River (Sulzfeld, Germany). Animals were in the range of 160–180 g when immunized and at least 1 week in the animal facility to get accustomed to the new environment. Rats were housed under standardized, pathogen-free conditions at the local animal facility (Medical Faculty, Ruhr-University Bochum, Bochum, Germany) where food and water were given ad libitum.
For immunization, 10 mg/kg xylazine (Xylavet, CP-Pharma, Burgdorf, Germany) and 50 mg/kg ketamine (CP-Pharma, Burgdorf, Germany) were used intraperitoneally (i.p.) to anesthetize the rats. Two hundred fifty microgram P253–78 in phosphate buffer saline (PBS) emulsified in equal volume of CFA containing 1 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, USA) were used to subcutaneously inject into the tail base of the rats. From the day of immunization, animals were weighed and scored daily. To determine a disease score, a tenfold system was used (0 normal; 1 less lively; 2 impaired righting/limb tail; 3 absent righting; 4 ataxic gait, abnormal position; 5 mild paraparesis; 6 moderate paraparesis; 7 severe paraplegia; 8 tetraparesis; 9 moribund; 10 death) (Enders et al. 1998). Animal experiments were approved by the North-Rhine-Westphalia, Germany authorities (Az.: 84-02.04.2015.A420).
For immunization, 10 mg/kg xylazine (Xylavet, CP-Pharma, Burgdorf, Germany) and 50 mg/kg ketamine (CP-Pharma, Burgdorf, Germany) were used intraperitoneally (i.p.) to anesthetize the rats. Two hundred fifty microgram P253–78 in phosphate buffer saline (PBS) emulsified in equal volume of CFA containing 1 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, USA) were used to subcutaneously inject into the tail base of the rats. From the day of immunization, animals were weighed and scored daily. To determine a disease score, a tenfold system was used (0 normal; 1 less lively; 2 impaired righting/limb tail; 3 absent righting; 4 ataxic gait, abnormal position; 5 mild paraparesis; 6 moderate paraparesis; 7 severe paraplegia; 8 tetraparesis; 9 moribund; 10 death) (Enders et al. 1998). Animal experiments were approved by the North-Rhine-Westphalia, Germany authorities (Az.: 84-02.04.2015.A420).
4
Canine Unilateral Orchiectomy and Implant Removal
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All surgeries were performed by the senior author, unblinded to the grouping. Animals were hemicastrated five months (154 days) following treatment with the implant or placebo. Orchiectomy of the right testis was performed under general anesthesia with propofol 2–4 mg/kg (CP-Pharma, Burgdorf, Germany) and ketamine 1 mg/kg (CP-Pharma, Burgdorf, Germany), after premedication with dexmedetomidine 5 µg/kg (CP-Pharma, Burgdorf, Germany) and levomethadone 0.2 mg/kg (MSD, München, Germany) maintained by isoflurane (1%) and following general surgical principles. The testes were immediately processed within the next minutes after surgery. After removal of the respective testis, the paraumbilical area was surgically prepared (scrubbed, disinfected) at the location of the implant. Following preparation, a skin incision was made where the implant could be palpated, and the implant was carefully surgically removed in total. In the case that the implant broke, specific care was taken to ascertain complete removal of all pieces. Afterwards, the previous implantation site was flushed with sterile saline. The incision was closed with a skin suture using Vicryl 3/0 (Ethicon, Norderstedt, Germany). All dogs received 4.0 mg/kg carprofen (Rimadyl®, Zoetis Deutschland GmbH, Berlin, Germany) for three days.
5
Histological Assessment of Testis Samples
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On D149, the left (second) testis was surgically removed under general anesthesia in all dogs by the senior author. The dogs were premedicated with dexmedetomidine 5 µg/kg (CP-Pharma, Burgdorf, Germany) and levomethadone 0.2 mg/kg (MSD, München, Germany), followed by the administration of propofol 2–4 mg/kg (CP-Pharma, Burgdorf, Germany), and ketamine 1 mg/kg (CP-Pharma, Burgdorf, Germany) maintained by isoflurane (1%). Immediately after surgery (D149), the testes were trimmed in pieces of approximately 1 cm3 and fixed in Bouin’s solution. Following washing with 70% ethanol and embedding in paraffin, the sections were cut, hematoxylin–eosin stained, and mounted with Roti® Histokitt (Carl Roth, Karlsruhe, Germany). A histological assessment of spermatogenesis was performed at 400× magnification (Zeiss West, Oberkochen, Germany [20 (link)]).
6
EAN Induction with P2 Peptide in Rats
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In the therapeutic setting, EAN was induced with neuritogenic P2 peptide, corresponding to the amino acids 53–78 of rat myelin P2 protein, synthesized by Dr Rudolf Volkmer from Charité University-Hospital Berlin, Germany. P2 peptide (250 µg) was emulsified in complete Freund’s adjuvants containing 1 mg/ml Mycobacterium tuberculosis H37RA (Difco) and injected s.c. into the tail base under anaesthesia with xylazine and ketamine (CP-Pharma). Disease onset was daily assessed by a blinded investigator using the following EAN score system: 0 = normal; 1 = less lively; 2 = impaired righting/limb tail; 3 = absent righting; 4 = ataxic gait/abnormal position; 5 = mild paraparesis; 6 = moderate paraparesis; 7 = severe paraplegia; 8 = tetraparesis; 9 = moribund; 10 = death.19 (link) With scores ≥7, the animals were euthanized from animal welfare measures.
7
Aortic Root Histopathology in Mice
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Mice were sacrificed after 25 weeks of treatment (at 45 weeks of age) by deep sedation (Ketamine (CP Pharma, Burgdorf, Germany)/Xylazine (Alvetra, Neumuenster, Germany), intraperitoneal) and exsanguination while blood was collected from the inferior vena cava. The animals were perfused via left ventricle with 10 mL phosphate-buffered saline solution followed by resection of the aorta. Then, the mice were perfused with 4% buffered formalin for paraffin sections of the aortic root. The heart was dissected from each animal, and the aortic sinuses were embedded in paraffin followed by serial sectioning (5 μm). Every third section was stained with a modified Movat’s pentachrome stain.19 (link)
8
Transcardial Perfusion and Brain Sectioning
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The animals were killed 13 days after surgical interventions. After anesthesia with a mixture of 1 ml/kg body weight ketamine (100 mg/ml; CP-Pharma) and 0.6 ml/kg body weight xylazin (20 mg/ml; CP-Pharma) delivered intramuscularly, rats were perfused transcardially with 0.9% saline followed by 300 ml of 4% paraformaldehyde, pH 7.4. The brains were removed, post-fixed overnight in a fresh solution, then cryoprotected with 20% sucrose for 24 h, at 4°C. Fifty μ m thick serial coronal sections were cut from the hypothalamus to the spinal cord on a Frigomobile (Frigomobil, Reichert-Jung, Vienna, Austria), and the sections were used for immunohistochemistry and immunofluorescence visualizations of BDA-labeling.
9
Murine Model of Pneumococcal Pneumonia
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Mice were orotracheally infected with S. pneumoniae (~6 x 106 CFU/mouse) as published elsewhere [73 (link),75 (link)]. In brief, mice received a short anesthesia with xylazine (3 mg/kg, Bayer, Leverkusen, Germany) and ketamine (75 mg/kg, CP-Pharma, Burgdorf, Germany). Subsequently, mice were intubated with a 26-gauge Abbocath catheter (Abbott, Wiesbaden, Germany) and the bacterial suspension (in 50 μl PBS) was instilled into the lungs under visual control. Following infection mice were brought back to their cages with free access to food and water. Mice were monitored twice daily for disease symptoms on the basis of an established scoring system and were euthanized according to previously defined criteria.
10
Postoperative Analgesia and Radiographic Monitoring
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All animals received postoperative oral analgesia with tramadol 0.5 mg/mL (Tramal, Grünenthal GmbH, Aachen, Germany) for five days.
In the first five days following the operation, the animals were checked daily, and afterwards 2–3 times weekly, concerning their weight, general behavior, gait and wound healing. On day 0, 7, 28 and 56, radiographs were taken under inhalation anesthesia.
After 56 days the rats were sacrificed by final cardiac puncture after an intraperitoneal injection of 60 mg/kg ketamine (ketamine 10%, cp pharma, Burgdorf, Germany) and 0.3 mg/kg medetomidine (Cepetor, cp pharma, Burgdorf, Germany).
In the first five days following the operation, the animals were checked daily, and afterwards 2–3 times weekly, concerning their weight, general behavior, gait and wound healing. On day 0, 7, 28 and 56, radiographs were taken under inhalation anesthesia.
After 56 days the rats were sacrificed by final cardiac puncture after an intraperitoneal injection of 60 mg/kg ketamine (ketamine 10%, cp pharma, Burgdorf, Germany) and 0.3 mg/kg medetomidine (Cepetor, cp pharma, Burgdorf, Germany).
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