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A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al., 2005, Gensel et al., 2006, Anderson et al., 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al., 2007, Gensel et al., 2006, Ballerman et al., 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al., 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement. The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique. The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.

. Recommended room layout for IBB behavioral testing (a) filming the animal within a perspex/plexiglass cylinder (b) filming the animal within its home cage.

B. ASSESSMENT OF VIDEOS:
Assessment of the animal's forelimb function should commence as soon as the animal starts to eat the cereal and stop as soon as the cereal piece is eaten.
Assessments should only be made with spherical-and doughnut-shaped cereals that are of a consistent size and shape prior to the initiation of eating. Animals should not be scored when eating pieces of cereal that are broken prior to testing.
For assessment, it is strongly advised that the speed of the videos be slowed down to at least half the original speed as even when injured, rats can eat quickly and the incidence of key behaviors may be missed. This can be achieved in programs such as QuickTime and Windows media player. Recently injured (i.e. 1-14 days post-injury), rats will take considerably longer to eat the cereal than rats at a later stage in their recovery. Thus, experimenters should not be concerned about the difference in the length of the video they use to assess the rat at early versus later stages in their recovery.
While observing the animal, use the IBB score sheet ( Figure 2) to note the presence of the following behavioral features. Recovery of forelimb function occurs from left to right of the IBB score sheet. The scoring sheet with individual categories that accompanies the Irvine, Beatties and Bresnahan forelimb scale. The first half of the sheet represents recovery of proximal forelimb function and the latter part of the sheet focuses on recovery of the forepaw.

Predominant Elbow Joint Position:
The rat is assessed for the most common position (more than 50% of the time) assumed by the elbow during the eating phase ( Figure 3).

SCORED AS:
• EXTENDED -The elbow is held straight with an angle of more than 160°.
• PARTIALLY FLEXED -The elbow is flexed with an angle of less than 160° but more than 90°.
• FULLY FLEXED -The elbow is flexed with an angle of less than 90°. The rat is assessed for the most common position (more than 50% of the time) assumed by the elbow during eating. Extended is when the elbow is held straight with an angle of more than 160°. Partially Flexed -The elbow is flexed with an angle of less than 160° but more than 90°. Fully Flexed -The elbow is flexed with an angle of less than 90°.

Proximal Forelimb Movements:
The rat is assessed for movements made by the shoulder and/or elbow of the impaired forelimb that may or may not result in contact of the forelimb with the cereal (Figure 4). [The rats are scored on the greatest extent of recovery they display during eating of the cereal. If animals show both slight and extensive proximal forelimb movements during eating they are scored as having extensive movements.]

SCORED AS:
• NONE -There are no shoulder and/or elbow movements of the impaired forelimb.
• SLIGHT -is defined as movements through less than a third the range of the shoulder and/or elbow joint. Twitches and shrugs fall into this category. • EXTENSIVE -is defined as movements that are more than a third the range of the shoulder and/or elbow joint. In early recovery, these movements can be numerous and erratic. The rat is assessed for movements made by the shoulder and/or elbow of the impaired forelimb that may or may not result in contact of the forelimb with the cereal. These proximal forelimb movements are defined as either: None -There are no shoulder and/or elbow movements of the impaired forelimb. Slight -is defined as movements through less than half the range of the shoulder and/or elbow joint, twitches and shrugs fall into this category. Extensive -is defined as movements that are more than half the range of the shoulder and/or elbow joint, in early recovery, these movements can be numerous and erratic.

Contact Non-Volar Support:
The rat is assessed for its ability to use the non-volar surface of the impaired forelimb to stabilize the cereal piece and in doing so, maintaining it in a position to aid eating. Areas of the forelimb that may act as supports are the forearm above the wrist, the wrist or the back of digits ( Figure  5).
* NOTE: Volar comes from the Latin word "vola" which the ancient Romans used for "the palm of the hand and the sole of the foot." SCORED AS: • NONE -No non-volar support by the forelimb during the eating phase. (less than 5% of the time) • SOME -Non-volar support of the object does occur during the eating phase but not always (less than 95% of the time).
• ALMOST ALWAYS -Non-volar support of the object occurs nearly always or always during the eating phase (95-100% of the time).

Figure 5. Contact Non-Volar Support.
The rat is assessed for its ability to use the non-volar surface of the impaired forelimb to stabilize the cereal piece and in doing so, maintaining it in a position to aid eating. Areas of the forelimb that may act as supports are the forearm above the wrist, the wrist or the back of digits. Contact non-volar support can be defined as either: None -No non-volar support by the forelimb during eating (<5% of the time). Some -Non-volar support of the object does occur during eating but not always. Almost always -Non-volar support of the object occurs nearly always or always during eating (>95% of the time). Non-volar support includes areas such as (i) above the wrist, (ii) the wrist or (iii) the back of digits.

Predominant Forepaw Position:
The rat is assessed for the most common position (more than 50% of the time) assumed by the digits, from flexed to extended, during eating ( Figure 6).

SCORED AS:
• CLUBBED, FIXED and FLEXED -The digits are flexed and held in a fist with joint angles of ~ 90°.
• EXTENDED NON-ADAPTABLE -The digits are extended with an angle of > 160° and do not conform to the shape of the held object.
• PARTIALLY FLEXED ADAPTABLE -The digits are partially flexed and conform to the shape of the object.

Contact Volar Support:
The rat is assessed for its ability to use the volar surface of the impaired forepaw to stabilize the cereal piece and in doing so, maintaining it in a position to aid eating (Figure 7).

SCORED AS:
• NONE -No support with the volar surface of the forepaw during eating (less than 5% of the time).
• SOME -Support of the object with the volar surface of the forepaw does occur during eating but not always (less than 95% of the time).
• ALMOST ALWAYS -Support of the object with the volar surface of the forepaw occurs nearly always or always during eating (95-100% of the time).
stereotypical depending on the size and shape of the cereal piece. The figures below depict the sequence of grasping techniques used by the unimpaired rat during consumption of the doughnut-shaped cereal ( Figure 12) and the spherical-shaped cereal ( Figure 13).

SCORED AS:
• ABNORMAL -Consistent use of an alternative method of grasping to the method used prior to injury to support and control the cereal piece during the eating phase. • SOMETIMES NORMAL -Inconsistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase. • ALMOST ALWAYS NORMAL -Consistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase. Figure 11. Grasping Methods. The rat is assessed for the most common (more than 50% of the time) grasping technique used during the eating phase and if this predominant grasping techinique is consistent with the technique used prior to injury. Several grasping methods exist but the most common are the (i) "precision", (ii) the "hook" and (iii) the "whole" grasp. The grasping techniques used by the rat are stereotypical depending on the size and shape of the cereal piece (See figures 12 and 13). Grasping methods used by the rat can be defined as Abnormal -Consistent use of an alternative method of grasping to the method used prior to injury to support and control the cereal piece during the eating phase. Sometimes normal -Inconsistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase. Almost always normal -Consistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase.

What if an animal has both contact non-volar and volar support?
If the animal is supporting the cereal by both non-volar and volar areas of the forepaw then that animal should be scored as having some contact non-volar and volar support.
In general the rat has subtle cereal adjustments, yet there is a single incidence of an exaggerated adjustment. How should I rate that animal?
As you move to the right of the sheet, the rat is penalized more heavily for the presence of abnormal behaviors no matter how infrequently they present themselves during the eating phase. This is to ensure that the rat is given a score that represents its best performance. Normal animals do not exhibit exaggerated cereal adjustments. In this case, the rat should be scored as having exaggerated cereal adjustments.

What does the term extended, non-adaptable paw position mean?
The digits of the forepaw are outstretched at an angle of 160 degrees or more, and do not conforming to the shape/size of the cereal piece.

How do I determine if the wrist joint is moving?
When establishing whether the wrist of the animal is moving during the eating phase, the observer should confirm that movements of the elbow and/or shoulder are not giving the appearance of wrist movements.

Can the wrist move prior to volar support?
Following extensive analysis of rats following varying extents of injury, wrist movement was, in general, observed once volar contact was made with the cereal.

How do I know if the rat is using a "normal" grasping method?
As with all behavioral tests, baseline data must be taken to establish how the animal grasps the object prior to injury. This is necessary as the animal may adopt an alternative grasping strategy following injury depending on the shape of the object. Following extensive analysis of rats prior to cervical injury we found that they use stereotypical grasping techniques depending on the shape of the object (as described in Figures 12  and 13). For example, with a sphere, the rat will generally start with a "whole grip" and then finish by using the "pincer grip", thus anything used in place of these grasping patterns would be considered abnormal.

How should I assign an IBB score?
The IBB is a 10-point ordinal scale of recovery progression with scores ranging from 0 to 9. Rats must fulfil all of the behaviors that describe each point or the rat is scored the closest deficit score.

Discussion
The Irvine, Beatties and Bresnahan forelimb scale (IBB) is a 10-point ordinal scale of forelimb recovery progression. The IBB analyzes many features of both proximal and distal forelimb use during the consumption of two whole cereal pieces set for the task, a spherical-and a doughnutshaped cereal. Videos of animals eating the cereals are evaluated for the most common forelimb behaviors (including joint position, object support, digit movement and grasping technique) used for both shapes and then an IBB score is allocated based on a combination of these behaviors selected to represent the greatest amount of forelimb recovery. Assessments should only be made with cereals that are of a consistent size and shape; thus, animals should not be scored when eating broken pieces of cereal. Animals must fulfil all of the behaviours that describe each point or the animal is scored the closest deficit point.
The IBB was created for assessment of forelimb recovery following experimentally induced cervical SCI. Initial studies in our lab found that, as with other behavioral assays, recovery assessed using the IBB score is sensitive to injury severity. The use of the IBB may not be limited to cervical SCI but may also be suitable for other forms of central and peripheral nerve injury that affect proper forelimb function.

Disclosures
No conflicts of interest declared.