Lab 2: Spinal Cord

Lab Summary

The purpose of this laboratory is to learn the gross and microscopic organization of the spinal cord and its functional components. Besides the study of the normal cord, there will be lesioned material from humans and animals that will be used to demonstrate certain neural tracts that have degenerated. You will also have the opportunity to compare the appearance of the cord stained by several methods that will emphasize different structures. 

SECTION 1: Gross Inspection of Spinal Cord 

SECTION 2: Microscopic Analysis of the Spinal Cord 

SECTION 3: Functional Analysis – Sensory Systems 

SECTION 4: Functional Analysis – Motor Systems 

SECTION 5: Meninges and Blood Supply 

SECTION 6: Review

Gross Inspection of The Spinal Cord

Human Spinal Cord Preparation 

Examine the gross dissections showing the surface features of the spinal cord and spinal nerves (Figure 2.1A-G).

During the lab session, there will be a human spinal cord, a full size plastic model of the vertebrae and spinal cord in each room and the AR Brain images of the spinal cord and nerves.

Review the surface features. Identify the dura, arachnoid and pia. Locate the dorsal root ganglia (DRG). The dorsal roots contain axons carrying sensory information into the spinal cord. Notice the division of the dorsal roots into a continuum of caudal to rostral rootlets, as they enter the cord at the posterolateral sulcus.

Your laboratory instructor will show you a horizontally cut segment of spinal cord in which you will be able to distinguish the gray and white matter, and the central canal. Finally, notice that the length of the roots increases at more caudal levels of the cord. Why is this?

You will also notice that the spinal cord is larger in the lumbar and cervical regions. What is the structural and functional basis for the lumbar and cervical cord enlargements?

In the sections of spinal cord that you will examine in this lab try to identify the posterior (dorsal) median sulcus, anterior (ventral) median fissure, posterolateral sulcus (dorsal root entry zone), gracile fasciculus, anterior commissure of the spinal cord, posterior (dorsal) horn, anterior (ventral) horn and anterior (ventral) root exit zone (Figure 2.2).

Note that the anterior commissure is the area of white matter anterior to the central canal and central gray matter area that contains axons crossing the midline. Within the anterior commissure of the spinal cord, spinothalamic axons cross to the opposite side to eventually ascend to the thalamus in the anterolateral white matter. This region is also the area of crossing for axons of the anterior corticospinal tract. The landmarks you have identified here can be found at all levels of the spinal cord.

Unique to cervical sections of the cord you should also be able to identify the posterior intermediate sulcus and cuneate fasciculus. Why are the posterior intermediate sulcus and cuneate fasciculus not found below cervical levels of the cord?

Use the Loyez myelin stained spinal cord series and Nissl stained sections identify the gross features discussed above at each level of the cord (Figure 2.3 and Figure 2.4). Note the staining of the large anterior (ventral) horn motor neurons. These neurons will give rise to the motor axons of the anterior root that supply somatic muscle. You may also examine these features more fully using the virtual microscope slide of the six levels of the cord in Interactive 2.1. 

Microscopic Analysis of the Spinal Cord

Differentiate between the levels of the spinal cord (cervical, thoracic, lumbar, sacral, and coccygeal) and explain the basis for their gross anatomical differences. 

Describe the function and modality of inputs and outputs for the different gray matter regions of the spinal cord, i.e. ventral, dorsal and lateral horns, as well as the intermediate gray area. 

Interactive 2.2 is a virtual microscope slide containing Kluver-Barrera stained sections of human spinal cord at cervical, thoracic, lumbar, and sacral levels. Myelinated axons are stained blue and cell bodies, nuclei and proximal dendrites are stained purple as seen in Figure 2.5.

Before microscopically examining each section of the cord, determine their orientation. For each section on Interactive 2.2, first distinguish between areas of gray vs. white matter; the gray matter has fewer myelinated nerve fibers and thus has a much lighter appearance.

Distinguish the posterior (dorsal) from the anterior (ventral) surface of each section (sections may not all be oriented the same way on the slides). Use the anterior median fissure, posterior median sulcus, and differences in the shape of the posterior and anterior gray and white matter areas as clues for determining the orientation of each section.

Compare the four sections on Interactive 2.2 and determine the approximate caudal to rostral level of the spinal cord that each represents. For clues, consider:

A) the shape of the gray matter, e.g., the cervical and lumbar enlargements have a larger anterior gray area that is expanded laterally for innervation of the limbs.

B) the relative amounts of white matter  relative to gray because of the accumulation of axons in ascending tracts related to more inferior (caudal) areas of the cord and the greater number of axons in the  descending tracts since few axons have yet to leave these tracts at cervical levels.

C) the size and shape of the cord e.g., the upper cervical, thoracic and sacral sections of the cord are more rounded, while the inferior cervical and lumbar regions are more elliptical.  

The general features of the spinal cord are most easily studied in the cervical and thoracic sections of the cord.

Under low magnification in Interactive 2.2 locate the posterior and anterior surfaces. Increase the magnification to examine which structures are stained. In the white matter, myelin is stained a sky blue. Select an area of ‘white matter’ and increase the magnification. Notice that the myelin (dark blue) forms a sheath around the axon, which is unstained; thus each nerve fiber (axon plus myelin) appears as a ‘doughnut’ in cross section.

The neurons have been counterstained violet with a ‘Nissl’ stain that stains RNA in the cell body and proximal dendrites. Neurons are found within the gray matter areas, are variable in size and tend to be the larger cells. The neurons generally have a rim of punctate purple staining of the cytoplasm and a paler nuclear area.

Glial nuclei and glial proximal processes are also stained. Within both the gray and white matter areas small deeply purple staining cells are the somas of oligodendrocytes forming the myelin wrappings of axons. The slightly larger cells that are paler staining and tend to have a more uniform purple staining than the neurons are astrocytes.

Much of the ‘neuropil’ is unstained and is composed of unmyelinated or lightly myelinated axons, small dendritic processes and synaptic terminals that contain little or no ‘Nissl’ substance. The cellular elements that fill these ’empty’ spaces can only be seen using other stains, as you will see later.

After grossly subdividing the gray matter into posterior (dorsal) horn, anterior (ventral) horn and intermediate gray, try to further divide these areas into individual nuclei and to visualize the Rexed’s laminae as illustrated in Figure 2.6.  

Substantia Gelatinosa (Laminae II): Almost always easy to find, even on gross inspection of fresh, stained or unstained material. The area is characterized by many small cells with little cytoplasm and by an absence of large myelinated axons, except for a few bundles of fibers passing ventrally. The limited number of large myelinated axons in this area gives it a pale appearance, particularly in myelin stained tissue. The area has the shape of an inverted ‘C’. Neurons in this area give rise to axons forming the spinothalamic tract and act as interneurons projecting to layer 1. They receive input primarily from C (unmyelinated) and A delta (thinly myelinated) fibers. The C fibers convey information for poorly localized and slowly conducting pain, while the A delta axons carry ‘fast’, acute pain information, as well as information related to temperature sensation, especially for cold. 

Nucleus Proprius (Laminae III-IV): Immediately ventral to the substantia gelatinosa, sits the nucleus Proprius. This area has both large and small neurons and many more myelinated fibers that are present both in bundles and scattered individually to give the appearance of a matrix. It receives inputs from the dorsal root ganglia that carry sensory information, such as light touch, touch and proprioception as well as some pain and temperature information. Cells in this area project to other laminae of the spinal cord, to the posterior column nuclei, and to supraspinal relay centers. 

Intermediate Gray (Lamina VII): Consists of small cells at the base of the posterior (dorsal) horn, as well as distinct nuclei that vary with the level of the cord. These include: l) the dorsal nucleus of Clarke, a group of large cells that forms a medial bulge and extends from Tl through L2; 2) the intermediolateral nucleus, forming a lateral horn in thoracic and upper lumbar levels; 3) the intermediomedial nucleus near the central canal, extending the length of the spinal cord; and 4) the sacral autonomic nucleus found at sacral levels of the cord. The dorsal nucleus of Clarke gives rise to the dorsal spinal cerebellar tract (DSCT), the intermediolateral and intermediomedial cell columns contain autonomic motor neurons of the sympathetic system projecting to sympathetic ganglia in the periphery and the sacral autonomic nucleus contains autonomic motor neurons of the parasympathetic system that innervate parasympathetic ganglia in the periphery. 

Using the what you have learned about the differences in the internal and histological organization of the different levels of the spinal cord, review the Loyez (myelin) and Nissl stained sections in Interactive 2.3 and try to determine which sections are from cervical, thoracic, lumbar and sacral levels of the cord. Identify the unique histological and nuclear features for each level and discuss them with your lab instructor to confirm your observations. 

Finally, draw the various nuclei and laminae on the spinal cord outlines in the Interactive 2.4.

Functional Analysis – Sensory Systems

Primary Afferents:

The first order neuron (“primary afferent”) for each sensory pathway entering the spinal cord is located within the dorsal root ganglion. These neurons have no dendrites within the ganglion and their single axon bifurcates, sending one process peripherally and one centrally.

The peripheral process is distributed distally to innervate the sensory receptors (or end as free nerve endings) in the dermatomes, as well as in the viscera. The central process enters the cord as part of the dorsal root and then branches extensively to project to nuclei of the spinal cord and brainstem (Figure 2.7). Prior to entering the cord the dorsal root divides into dorsal rootlets, the majority of which enter the cord at the posterolateral sulcus.  

The axons of primary afferents are of many diameters, the smallest are unmyelinated. Classes of large myelinated axons convey impulses from muscles, tendons, joints, and cutaneous touch receptors; most small myelinated and unmyelinated axons convey impulses from nociceptive, pain or temperature receptors. 

On Interactive 2.1 and 2.2 and 2.8 examine the dorsal roots of any section at high magnification and note the variable diameters of the component axons. Study the dorsal root entry zone. The majority of large, myelinated axons entering at the posterolateral sulcus travel to the dorsal white matter forming the posterior (dorsal) columns.

What functional modalities of sensory information are typically conveyed by fibers of different size and myelination? 

Major Ascending Systems:

Return to Loyez myelin stained spinal cord series. At each of the spinal levels on this slide, consider the location of the major ascending tracts of the spinal cord and their cells of origin. Those cells that receive direct input from the primary afferents (dorsal root axons) and project to the brain are often called secondary afferents or second order neurons.

The boundaries of the individual tracts, except for the posterior (dorsal) columns, are not visible within the white matter. At this point, however, it is important to learn their relative location, as this will be of significant diagnostic value in locating spinal lesions.

Examine the sections above and below a human spinal cord transection in Figure 2.9. Note the areas of axonal and myelin degeneration in the section superior to the transection. The regions normally occupied by ascendin tracts are visible due to degeneration of the constituent axons resulting from their separation from the neuron body at lower levels of the cord. These areas appear pale staining in portions of the white matter that normally would be darkly stained.

Based upon your knowledge of where ascending and descending pathways would be found in the white matter which section is likely to be above the level of the transection and which below in Figure 2.9? Why does separation from the soma result in axonal degeneration in these areas?

Spinothalamic Tract / Anterolateral System (Figure 2.10A):

The first order neuron for this ascending pathway is found in the dorsal root ganglion (DRG). Axons enter the cord as the dorsal roots, many entering as fibers of the Tract of Lissauer. Lissauer’s tract is a small group of unmyelinated or lightly myelinated axons that cap the posterior horn and carry pain and temperature information. The second order neurons for this system are found at all spinal levels in laminae I, II & V (the posteromarginal nucleus, substantia gelatinosa and the neck of the posterior (dorsal) horn). Axons of these cells cross the midline ventral to the central canal and ascend in the anterolateral (ventrolateral) white matter of the contralateral side. These crossed axons then terminate in areas of the brain stem and thalamus.

Function: Relay of pain, temperature and some touch sensation to the thalamus. 

Posterior (Dorsal) Columns (Figure 2.10B):

The first order neuron for this ascending pathway is found in the dorsal root ganglion. Axons enter the cord as the dorsal roots, most entering medial to the Tract of Lissauer and the posterior (dorsal) horn. These axons of first order dorsal root ganglion cells (primary afferents) remain uncrossed and ascend within the posterior (dorsal) columns. Within the posterior columns the more medial fibers carry sensory information from the lower part of the body. Axons from more superior regions of the body are situated more laterally in the dorsal columns. At cervical levels of the cord the posterior intermediate sulcus forms in the dorsal columns and separates axons in the more lateral portions of the column which are from the upper trunk, arms and neck fasciculus cuneatus, from the more medial axons that are from the legs and lower trunk, the fasciculus gracilis. As the axons of the posterior columns reach the caudal portions of the medulla they terminate on second order neurons in the posterior (dorsal) column nuclei. Axons that ascended in the fasciculus gracilis terminate in the nucleus gracilis and axons of the fasciculus cuneatus terminate in the cuneate nucleus. 

Function: Relay of touch (flutter, vibration), pressure and position sense information to the posterior (dorsal) column nuclei, which then project to the contralateral thalamus.

Functional Analysis – Motor Systems

Motor Neurons:

Examine the different levels of the spinal cord in Interactive 2.2 and in the Loyez myelin stained spinal cord series (Figure 2.3). Consider the presence or absence of the three main groups of motor neurons: 

The most medial somatic motor neuron pools innervate the axial muscles (i.e., the postural muscles of the trunk) and are found closest to the midline in the anterior (ventral) horn throughout the length of the cord (Figure 2.12 and Figure 2.13).

Motor neurons that innervate the more distal musculature are found progressively more laterally in the ventral horn. For example, the muscles of the shoulders and pelvis, at cervical and lumbar levels would be found lateral to those innervating the axial musculature. The motor neurons innervating the proximal muscles of the arm (or leg) would be next and finally, motor neuron pools that innervate the distal parts of the extremities, the fingers or toes, would lie farthest from the midline. The lateral motor neuron pool is especially large at the levels of the cervical and lumbar enlargements.

What is the arrangement of cells that innervate flexors vs. extensors, and the distal vs. the proximal portions of the limbs (Figure 2.13)? 

Visceral motor neurons (Figure 2.12). Neurons of the intermediolateral nucleus are found from C8 to L3, are preganglionic cells of the sympathetic nervous system, and terminate in the sympathetic ganglia. Neurons of the sacral autonomic nucleus are preganglionic cells of the parasympathetic nervous system and terminate in visceral ganglia. Both groups of axons are located at the lateral border of the intermediate gray. 

Note the exiting axons of the motor neurons in the anterior (ventral) gray, and use Interactive 2.1 and 2.8 to examine cross-sections of the ventral roots at at high magnification. In contrast to the variety of axon diameters found in the dorsal root, only two basic sizes of myelinated axons are found in the ventral root. The large axons arise from alpha motor neurons, the smaller are from gamma motor neurons. What type of muscle fiber does each supply? Recall that up to 30% of the axons in the ventral root are unmyelinated and small myelinated afferents from the dorsal ganglion. 

Major Descending Spinal Tracts:

In examining the spinal cord sections, consider the locations of the major descending tracts. As in the case of the ascending systems, the descending tracts are not clearly visible in sections of normal cord. After reviewing the characteristics of the major tracts below, you will re-examine the human spinal cord transection slide in which some of these tracts will be visible because they have degenerated following transection of the cord (Figure 2.9). 

In general, there is a topographical mapping of the position of descending tracts in the spinal cord white matter and the motor neuron pools they innervate (Figure 2.13). Laterally located tracts (lateral corticospinal and rubrospinal) project to lateral motor neuron pools and have their primary influence on specific volitional movements of distal limbs. Medially located tracts (reticulospinal, vestibulospinal and tectospinal) project to medial motor neuron pools and predominantly influence neck, trunk, shoulder and hip anti-gravitational movements.

Tracts Predominately Controlling Distal Limb Muscles:

Lateral Corticospinal (Figure 2.14A):

This tract originates from neurons of the motor and sensory areas of the cerebral cortex. It descends in the internal capsule, to the cerebral peduncles and then passes through the anterior pons and medulla. It crosses to the opposite side of the brain near the medullary-spinal cord junction after which it descends in the lateral funiculus (white matter) before eventually innervating motor neurons and interneurons in the anterior horn for all levels of the spinal cord. The primary site of termination for these axons is on interneurons in laminae VIII of the anterior horn that then project onto the motor neurons in the lateral motor neuron groups of laminae IX. Although some axons of the corticospinal tract end directly on motor neurons, this is uncommon. This tract is crossed and travels in the posterior-lateral white matter of the cord.

Function: Its principal action is to control movements of the extremities. It is essential for the dexterity and precision of movements of the digits and joints. 

Rubrospinal (Figure 2.14B):

This tract originates in the red nucleus of the midbrain and the axons immediately cross to the opposite side of the brain in the anterior tegmental area of the midbrain. The axons then travel in a lateral position in the pons, medulla and spinal cord before innervating lateral motor systems throughout the cord. As for the corticospinal axons, the rubrospinal axons terminate on interneurons and motor neurons of the anterior horn. This crossed tract travels in the lateral white matter of the cord just anterior to the lateral corticospinal tract.

Function: It is involved in the control of movement for the contralateral limbs although it is less important than the lateral corticospinal tract for these actions in humans. Activation causes excitation of flexor muscles and inhibition of extensor muscles. Although the function of this tract in humans is unclear it is thought to be involved in movement velocity, as lesions cause a temporary slowness in movement; likely plays a role mediating learned motor commands in coordination with its’ cerebellar connections; and most likely is an important pathway for the recovery of some voluntary motor function after damage to the corticospinal tract. 

Tracts Controlling Trunk and Proximal Limb Muscles 

Axons of these tracts are found predominately in the anterior medial portion of the spinal cord white matter, the anterior funiculus. 

Anterior Corticospinal (Figure 2.15A):

Axons of this tract originate from neurons of the motor and sensory areas of the cerebral cortex, and descend with the lateral corticospinal tract in the internal capsule, the pons and medulla until reaching the caudal medulla where the lateral corticospinal axons decusate. Axons of the anterior corticospinal tract do not cross at the medullary-spinal cord junction. They remain on the ipsilateral side traveling in the anterior medial white matter of the spinal cord to innervate medial motor neuron pools and interneurons in the anterior horn at all levels of the spinal cord. The primary site of termination for these axons is on interneurons in laminae VIII of the anterior horn, that then project onto motor neurons in the medial pools of laminae IX. Although some axons of the corticospinal tract end directly on motor neurons, this is uncommon. This tract is uncrossed as it travels in the anterior-medial white matter of the cord. At the level of innervation of the motor neurons in the anterior horn most of these axons cross prior to entering the anterior horn, although some remain uncrossed.

Function: Control of the axial and girdle muscles.

Medullary and Pontine Reticulospinal (Figure 2.15B):

These tracts originate from the reticular nuclei in the medulla and pons and supply motor neurons throughout the cord. They descend as bilateral and uncrossed tracts, respectively, in the anterior white matter of the cord and terminate on motor neurons and interneurons in the medial portion of the anterior (ventral) horn.

Function: Control axial, proximal extremity and girdle musculature. They are involved in postural tone, balance and automatic gait related movements. 

Lateral Vestibulospinal (Figure 2.15C):

This tract originates from the lateral vestibular nucleus in the medulla and supplies motor neurons throughout the cord. This tract is uncrossed and is located in anterior (ventral) white matter.

Function: Control axial, proximal extremity and girdle musculature. Involved in postural tone and balance. 

Tracts Controlling Head and Upper Trunk

Tectospinal (Figure 2.16A):

Originates from the superior colliculus of the midbrain, it crosses to the opposite side of the brain in the midbrain and descends to terminate in upper cervical segments of the cord. This tract descends in the ventromedial white matter of the cord.

Function: Mediates reflex postural movements in response to visual, auditory and somatosensory stimuli and is involved in the coordination of head and eye movements. 

Medial Vestibulospinal (Figure 2.16B):

A component of the medial longitudinal fasciculus (MLF). Originates from the medial vestibular nucleus in the medulla and supplies the medial motor horn areas of cervical and upper thoracic cord. This tract is predominately uncrossed and descends in the ventromedial white matter.

Function: Is involved in the positioning of the head and neck. 

Having reviewed the descending pathways, now examine the low cervical section from below the level of cord transection in the slide of the human spinal cord transection (Figure 2.17). Degenerating axons (lightly-stained areas) in this section arise from cell bodies above the transection, and the pattern of degeneration can be used to locate descending tracts of the cord. In the dorsolateral white region, the large pale area consists of degenerating corticospinal and rubrospinal axons. Although these two tracts overlap and cannot be distinguished in these sections, review Figure 2.18 to get a sense of their relative positions.

What is the somatotopic organization of axons in these tracts relative to the level of the cord that they will innervate? What groups of motor neurons or premotor neurons receive heavy input from these tracts? Are the tracts crossed or uncrossed?

The remaining descending tracts, i.e., the tectospinal, the vestibulospinal, and the reticulospinal, cannot be distinguished on this slide because of their intermingling with other fibers and their relatively small area.

In Figure 2.18 review the positions of the ascending and descending pathways of the spinal cord, noting whether the pathway is crossed or uncrossed, where it crosses, what is the source and modality of the sensory or motor information, the target motor neurons of the descending tracts and the function of each tract. 

For review, draw the ascending and descending tracts on the diagrams of transverse sections of the thoracic spinal cord in Interactive 2.7 You should also review the important features of these pathways using the Tables of Ascending Sensory Pathways and Descending Sensory and Motor Pathways (TABLE 2.2-2.3).

Meninges and Blood Supply

Use the labeled hematoxylin and eosin stained section of the rabbit spinal cord (Figure 2.19) and the virtual microscope hematoxylin and eosin stained sections (Interactive 2.8) to identify the dura and arachnoid tissues. The pia is difficult to see in these sections. In these sections identify the anterior (ventral) median fissure, the adjacent anterior spinal artery, and separately, near the dorsal root entry zone locate the posterior spinal arteries. In the upper cervical spinal cord and lower medulla the anterior spinal artery arises from the fusion of branches from each of the two vertebral arteries. The posterior spinal arteries arise from the posterior inferior cerebellar arteries (PICA). The vertebral arteries in turn arise from the proximal subclavian arteries. 

Review the principal arteries supplying the spinal cord and the supply of the internal spinal cord (Figure 2.20A-B). The anterior and posterior radicular arteries arise arise from segmental vessels (ascending cervical, deep cervical, intercostal, lumbar, sacral arteries) at every spinal level and serve their respective roots and ganglion. The anterior and posterior spinal medullary arteries (also called medullary feeder arteries or segmental medullary arteries) arise at intermittent levels and augment the descending flow in the anterior and posterior spinal arteries, respectively. The arterial vasocorona is a diffuse anastomotic plexus covering the cord surface.

Revisit these arteries on the anterior (ventral) surface of one of the whole brain specimens. The posterior and anterior spinal arteries continue along the longitudinal axis of the spinal cord forming a spinal arterial plexus that surrounds the cord. Their supply of blood is augmented by segmental medullary arteries arising from the aorta at various segmental levels.

A particularly prominent medullary artery found between T9 and T12 is called the Artery of Adamkiewicz (Figure 2.21). This artery provides the major blood supply to the spinal arterial plexus of the lumbar and sacral cords. The area of the cord between T1 and T4 is not directly supplied by a major medullary artery and is particularly vulnerable to infarction. These regions of the spinal cord receive blood supply from the most distal branches of the large arteries, and are called watershed areas. Due to there supply by the most distal branches of these arteries damage due to decreased aortic pressure or perfusion can be particularly problematic.  Sometimes the cord between T4-T6 is also included in this vulnerable region.

Review