**3.4 Determination of anxiety**

The test for anxiety was done using:

*Ameliorative Effects of Zinc and Vitamin E on Physiological Changes after Exposure to Heavy… DOI: http://dx.doi.org/10.5772/intechopen.111518*

### 1.Elevated Plus Maze (EPM)

### 2.Light and Dark Transition Box (LDTB)

### *3.4.1 Elevated plus maze (EPM)*

Rodents are used in the Elevated Plus Maze (EPM), a test for detecting anxiety in lab animals, as a general research tool in the study of neurobiological anxiety as well as a screening test for potential anxiolytic or anxiogenic substances. The animal in the EPM displays this concern by spending greater time in the enclosed arms [30, 31].

A raised, plus-shaped (+) apparatus with two open and two enclosed arms is used for the test. The behavioral model is based on rodents' typical dislike of open areas. Due to this aversion, a behavior known as thigmotaxis develops, which is the desire for staying in enclosed areas or close to the boundaries of a confined region. This results in the animals restricting their movement to the confined arms of the EPM. An increase in the proportion of time spent in the open arms (time in open arms/ total time in open or closed arms) and an increase in the proportion of entries into the open arms (entries into open arms/total entries into open or closed arms) are indicators of reduced anxiety in the plus maze. Occasionally, the total number of closed-arm entries is used as a gauge of overall activity [31, 32].

The EPM was created in accordance with Lister's specifications (1987). From a center square (5 x 5 cm), the maze contains two open arms (45 5 cm2 ) with 0.25 cm high borders and two closed arms (40 5 cm2 ) with 15 cm high walls. There is a small ledge in the open arms (4 mm high) to stop the mice from losing their footing and going off the edge. Because they are contained, like most anxiety tests, the closed arms give a feeling of security. This job takes advantage of mice's natural desire to investigate novel surroundings and their aversion to wide open spaces. Anxiety is also quantified by the open arm avoidance score [33].

To remove olfactory cues as well as feces and urine, the surfaces and closed sides of the plus maze arms were washed with methylated spirit prior to the test. The mouse was positioned in the plus maze's middle square so that it initially faced an open arm away from the experimenter. Mouse was given five minutes to examine the device after placement before a silent stopwatch was started. The testing procedure was documented. Open arm movements and head dipping were deemed exploratory behaviors, and a higher frequency of these actions indicates a higher level of investigation [34].

Following are the behaviors scored:


### *3.4.2 Light/Dark transition box (LDTB)*

Two compartments make up the LDTB device. The light compartment occupies two-thirds of the box and is both open and well-lit. A covered and dark compartment makes up one-third of the entire box. The two chambers are connected by a 7-cm door. Rodents choose shadowy environments than bright ones. Rodents, on the other hand, show a propensity to explore when placed in an unfamiliar habitat. There are visible indications of anxiety as a result of these two opposing feelings. The dark compartment is often where rodents spend more time than the bright one. The percentage of time spent in the light compartment will rise in animals given anxiolytic injections. Rearing, or when a rodent raises up on its hind legs, is an indication of motion and nighttime exploration increase in compartment as well. The amount of time spent in the dark compartment increases after receiving anxiogenic injections. There is no prerequisite training for the LDTB. No food or water is restricted and only natural stressors like as light are utilized [35, 36].

The wooden light/dark box (45 x 27 x 27 cm) has two compartments that are of different sizes. Two-fifths of the box is painted white for the bigger compartment (27 x 27 cm), while two-fifths of the box is painted black for the smaller compartment (18 x 27 cm). A door (7.5 x 7.5 cm) that is situated in the middle of the wall between the two compartments at floor level connects them. The Plexiglas-covered floor is separated into 9 x 9 cm squares. The covers of both sections are made of transparent Plexiglas.

The apparatus's light box is filled with a mouse, which is given free rein to move about. The mouse will typically explore the compartment's edges before discovering the door. The mouse is given five minutes to investigate the device, and the rodent's actions inside the box recorded. To be deemed an entry, all four paws must be inserted into the opposing chamber. The mouse is then taken out, and the box is cleaned with cotton wool and 70% ethyl alcohol. The box is then allowed to dry in between experiments [37].

*Ameliorative Effects of Zinc and Vitamin E on Physiological Changes after Exposure to Heavy… DOI: http://dx.doi.org/10.5772/intechopen.111518*

### *3.4.3 Behaviors score*

