Abstract

There are many publications that deal with administration of test substances to animals and removal of blood samples for different objectives. Parenteral route of drug administration includes further different routes i.e., drug administration through muscles (IM route) through veins (IV route) and through subcutaneous route. We can manage to check the outcomes of test substances within minutes when we go for IV route as in this route drug directly go to systemic circulation. Different experimental animals are utilized to check the possible outcomes of test substances that include Rabbit, mice, and rat. Various factors like dose strength, dose frequency and the meaning by which drug can be administered to animal can be examined. The emphasis on drug safety should be the main priority while injecting drug substances. Animal ethics guidelines should be followed in this regard. AEG basically provides us safety guidelines which should be followed for safety handling.

Key Words

Intramuscular, Anesthesia, Doses, Administration, Mice, Subcutaneous, Parenteral

Introduction

To meet our essential regulatory demands, experimental animals play a vital role. As their dosing is compulsory for several scientific investigations. To check the safety margins of new drugs, it is important to check the effective doses by having trials on experimental animals. When chemicals have low toxic properties or they are poorly soluble, we must use larger volumes to entertain scientific as well as regulatory requirements.

Now administering of different substances to experimental animals needs a lot of careful experience. The focus should be on maximum positive outcomes and minimum relative adverse events. There are a lot of routes to which a drug can be administered to the experimental animals, but our main focus would be on parenteral route of drug administration.

Parenteral route has further several routes by which drug would be administered. Intravenous route is that where drug is directly delivered to the blood by puncturing the vein of animal. Intra-arterial route involves drug delivery through arteries of animals. Intraperitoneal route is linked with drug delivery through abdominal cavity. Subcutaneous administration involves drug delivery through the skin of the animals. Now when we place the drug between the two layers of skin of animal it would be called intra-dermal route of drug administration when drug is delivered to muscles it would be intra-muscular route of drug administration. The most efficient way of drug delivery to animals is the intracutaneous route of drug delivery, as it avoids requirement of solute absorption. Here the drug is administrated in the form of bolus or infusion directly to the blood vessels. (Glenny, Buttle, & Stevens, 1931)

When we talk about pharmacokinetic studies and different pharmacokinetic properties in animals, the one route of drug administration that is widely used in mice and other rodents is the intraperitoneal route of drug administration. While in the case of rabbits, the most frequently used route is the intravenous route of drug administration. While we apply any of routes of drug administration, the focus should be on proper control of the animals, as these animals should be restrained efficiently to stop useless struggles by the animal. All the needles used should be sterile. The drug given to animal should not cause discomfort to the animal or any of the technique that cause harm or discomfort to the animal should be strictly prohibited. (Richard et al., 2011)

Factors Affecting Parenteral Route

When the drugs are administrated to animals from parenteral route, different factors play a vital role for positive output. These include volume of the dose used, checking the stability of substance, pH value, viscosity, osmolality studies, sterility studies of the formulation and biocompatibility studies. The needle size should be smallest in parenteral route for better outcomes.

Vehicles for Drug Administration

Selection of vehicle for drug administration in animals is an important factor. Vehicles should be selected that follow specific criteria i.e., it should not affect the biological activity of drugs, means it should be biologically inert. It should have no harmful effects on the animals.

Most widely used vehicles are some isotonic solutions, co-solvents, certain oils, and some buffered solutions. When we have to administer drugs in the form of suspensions, its viscosity and PH should be checked accurately. (Vajanto et al., 2002)

Requirements for parenteral administration

1. Animal restrained

2. Razor

3. Gloves

4. Warning lamps with bulbs of 40 to 100 watts

5. Needle of 27 to 30 gauge and 1 ml capacity for mouse

6. Needle of 22 to 25 gauge for rabbits

7. Needle of 20 gauge for rats

8. Alcohol pad

9. Cotton

Parenteral administration to rabbit

Intravenous administration

Here marginal ear vein of rabbit is used to deliver the drug substance. The procedure followed in this specific category is as follows:

1. Animal should be placed in restrainer carefully.

2. The hair over marginal ear vein is removed carefully.

3. Injection site at the ear should be cleaned with alcohol swab.

4. Now insert the needle with gauge of 22-30 size with syringe or here butterfly needle can also be used.

5. After inserting the syringe in the ear vein, pull the syringes plunger back with pressure. If there is flash of blood present, it shows that the syringe is not properly placed.

6. Now administer the drug substance in steady state motion.

7. Take an alcohol pad and apply it to the withdrawal site with pressure so that bleedizzng can stop.(Shyu, Manor, Magner, Yancopoulos, & Isner, 1998)

Now there are some considerations that are followed in IV administration of drug in rabbits, which includes this process has a rapid absorption rate. It can be easily performed by using general type of anesthesia. The volume should not be more than 1% of the total body weight of rabbit.

Intramuscular Injection to Rabbit

In this category, the drug substance is injected to the thigh muscles. This route of drug administration may be painful as muscle fibers got disturbed by drug substance material. There are high risks of nerve damage in this category, so site should be chosen with great care. Oily formulations can be injected by this route as these formulations remain in depot form in muscles for more than 24 hours. Candal injections should be avoided as they can damage the sciatic nerve. Now IM injection involves following steps:

1. Animal should be restrained manually.

2. Injection site should be cleaned by alcohol swab.

3. Needle should be cleaned and inserted in such a manner that it should not harm sciatic nerve.

4. The syringe plunger is pulled back with pressure, if there are some fleshes of blood, it shows improper placement.

5. The drug substance should be administered within an optimum range, as too rapid administration can lead to problems.

Subcutaneous Injections

These types of injections are injected in the loose skin near the neck region. It forms a space underlying between the muscles and the outer skin. This route of drug administration is not suitable for irritant drugs as it can lead to severe skin problems. This route is used commonly. It can be observed without the use of general anesthesia, but proper handling is very important for adequate administration of drug to animal. The needle should be placed at angle of 45^o. The process of injecting the drug material to animal should be smooth and there should be no harm to the animal in any of the way. Now the process for proper subcutaneous injection procedure involves following steps:

1. To restrain the animal is the most important of step in the subcutaneous administration of drug to animal. It should be restrained on table.

2. The injection site should be cleaned with alcohol pad.

3. Needle should be inserted near the skin of neck between outer skin and the muscles.

4. The plunger of syringe is pulled back with force, if there is any flesh of blood, needle is not properly maintained.

5. The drug substance should be administered in a steady motion.(Bonanomi, Velvart, & Weder, 1987)

Parenteral Routes in Mice

It mainly depends on the extent of response we want. The person performing the experiment should fully know

about the chemicals and vehicles he was using because the chemical may cause irritation and may be toxic for animal. The place where medicine should be injected should be determined accurately by bringing the plunger back if there is blood on pushing back then it is correctly placed and if there is fluid then there is error. Several parenteral routes are for the mice first is subcutaneous in which mice are put in normal restrainer or it is properly handled and with one hand skin pouch that is loose skin on the top is being stretched and injection is being placed.(Dupuis, McDonald, & Ott, 1999) Pellets are used for long term effect in case of intraperitoneal injection no anesthesia is required. One person holds the mice while other puts injection, so absorption is comparatively fast. Intravenous and intra-arterial route are commonly known as intravascular route. In case of these routes two veins are preferable tail vein and jugular vein. In tail vein anesthesia is not required while in jugular vein there is need of anesthesia. In case of irritating substance there is need that injection should be diluted prior to injecting. In case of retro orbital injection in mice anesthesia is administered. (Briner, Kuo, Keating, Rogers, & Greenstein, 1993)It is the most complicated type of route because it may lead to tumor so to avoid this they must be shake prior to administration. In this injection is administered to retro orbital sinus.(Xue et al., 2012) Next is intramuscular in which the injection is administered directly to muscles but in mice it is not administered due to lack of muscular mass and there are chances of sciatica if they are not administered properly in the gluteal muscles. In case of intradermal injection there is lack of inflammation and immunologic studies that is why it is not administered. If the safety policy approved, it then it is administered. In case of ocular injection, it is very limited and only use for certain compounds. There is need of intranasal injection and volume should not exceed from 30 microliter in one nostril. Another route is intra tracheal route in which mice is placed in vertical position and volume injected is 50 microliter and head are in upward direction with tongue out and the injection is aspirated. Next is intracranial injection in which either bolus is given or there is continuous administration. It can be done via surgery or direction injection. Next is intrathoracic administration in which injection is in thoracic cavity i.e. in chest with aid of anesthesia. (Israelit, Long, White, & Hull, 1973)

Injection Volume

Different Volume are administrated for different injections e.g., for subcutaneous injection the site is flank, or the skin mass is more and about 1-2ml is administered for the subcutaneous. Similarly in case of IM the injection is placed in gluteal muscles, perpendicular to the muscles and volume should be 0.03-0.05ml and in case of intra peritoneal injection as injection is placed in peritoneal cavity that in lower right quadrant so amount of injection is less than 2ml. Similarly, in case of IV injection the injection should be placed in lateral vein and in 1V infusion the solution is placed through cannula. In case of retro orbital injection should be placed in sinus with volume of 180- 200 ml with 29-gauge needles. In case of intra cranial injection it is placed in cranium with the help of cannula and its needle is not available. The parenteral route of administration had many advantages like they can be used for patients who are continuously vomiting. They can be used for substance having irritating taste. They also had very high bioavailability and rapid effect as compared to oral substances.(Barondes & Cohen, 1968)

Method of Administration Through different Route

First of all, mice should be handled properly, or they are kept in restrainer or cage. There should be proper lightening system. They should be left for something in order to get rid of anxiety then their tail is move around in order to see the vein properly. Once the vein is clearly seen then the needle is injected into the tail vein the plunger is pulled back. If there is blood, then needle is not placed properly, and second indication of improper placement is the production of bleb which ultimately leads to chronic disease. We can inject to other side if the needle is not at its correct position. We can inject at least five sites on tail vein once injection is placed properly then put out the needle and clean the site properly. In case of intramuscular injection, the injection is placed in gluteal or thigh muscle. For these mice should be hold after correctly holding the mice and then the injection is placed at muscle at 90° or 45° and we have to check if the injection is placed correctly by pulling the plunger back. In intra peritoneal injection the mice should be placed properly in resting position that is in restrainer or it can be hold by one personal and injection should be placed by other personal. (F. Wu et al., 2012)

For injecting intraperitoneal injection, the mice should be hold properly that is his head is facing in downward direction and injection is administered in parallel direction in abdominal region that is rich of blood vessel that is third quadrant. In order to check if the injection is properly placed or not the plunger is move back if there is blood on pushing back the plunger then it is not correctly placed. (Engineering, 1994)

Intracranial injection should be administered to mice in order to treat the brain tumor. The intracranial injection is injected through the post glenoid because this is most easy pathway for the entrance and the florescent technique is used to locate the tumor. This is most easy procedure for intracranial injection. Usually intradermal needle is used for this purpose.(Ledwith et al., 2000)

Intra thoracic injection can be mainly used to treat and locate lung cancer. First of all the mice is slightly bent and special kind of needle is used that is slightly curved and the injection is placed into the ribcage between two ribs and mainly in the middle and the most important thing to be considered is that it should not puncture the lung .

In case of intra nasal injection the mice are put in cage with face and toe in upward direction that is usually a supine position. It is administered with help of micropipette and injection is usually placed through this. But the main disadvantage is that mice may die due to suffocation.(H. Y. Wu, Nahm, Guo, Russell, & Briles, 1997)

Precautions

Certain points needed to be considered while injecting the most important is that there should be a proper restrainer in which the mouse is placed. Secondly injection may irritations so normal saline can be used to avoid this. All the apparatus being used during the experiment should be clean. Thermometer should be placed in order to check the temperature of the mice. Several precaution should be taken depending on type of injection e.g., in intravenous injection the vein should be dilated, intramuscular and intracranial injection should be properly placed to avoid the sciatica and tumor respectively. (Makino et al., 2012)

Conclusion

As we know that at present, there are a lot of methods by which blood can be removed from different species of animals in a better way. In the past blood removal from small rodents was not so easy as there were a lot of hurdles in blood removal. Some of methods of blood removal from different animals require specific considerations i.e., some methods require anesthesia for smooth removal of blood and at the same time there are a lot of side effects when we apply specific methods for blood removal from different animals for any scientific objective i.e., those side effects may be pain at injection site or stiffness of blood vessels. In this modern era where different scientific technologies and software are available, it is still a huge risk to check the outcomes of a novel drug by directly administering to humans Animal testing of drugs should be done by strictly following the animal ethics guidelines. If any injury happens to animals, proper treatment strategies should be applied. As we know cancer is the leading cause of death worldwide, so those drug bases cancer treatment strategies should be tested on animals to have best treatment outcomes on humans.

Blood volume circulating in laboratory animals (Blood volume in ml per kg)
Species	Recommended Mean	Range of means
Rabbit	56	44-70
Mice	72	63-80

Sites Suggested for Repeated Blood Sampling
Species	Suggested sites
Rabbit	Marginal ear, central ear artery and jugular vein
Mice	Saphenous or lateral tail

Route and volume (ml per kg)
Species	IP	IM	IV
Rabbit	5	0.25	2
Mice	20	0.05	5