Muscle Histology
Muscle types
Type I and II. Based on histochemical reaction for myosin adenosine triphosphatase (ATPase)
Type 1 muscle fibers: These are slow twitch, fatigue resistant, have slow oxidative metabolism (high number of mitochondria), and low glycolytic activity, stain lightly at alkaline pH (9.4) and darkly at acidic pH (4.6)
Axial muscles
High lipid content, low glycogen, low glycolytic enzyme content
They are red in color and small in diameter.
They have more capillaries than type 2 muscle fibers.
Axons that innervate these fibers are smallest and conduct slowly.
Type IIa muscle fibers: These are fast twitch, intermediate fatigue-resistant, fast oxidative and glycolytic metabolism, stain darkly at alkaline pH (9.4) and lightly at acidic pH (4.6)
Low lipid content, low number of mitochondria, high glycogen, high glycolytic enzyme content with high glycolytic capacity and moderate oxidative capacity.
They are red in color and large in diameter.
Type 2 fibers are twice as many as Type 1 fibers
Type IIb muscle fibers:
Fast twitch, poor fatigue resistance, fast glycolytic metabolism
Their color is white and diameter is large.
Axons that innervate these fibers are large and conduct fast.
Type IIc muscle fibers are undifferentiated and embryonic type. Comprise only 1-2% in adults.
Will be strongly reactive at both acid and basic pH to ATPase myofibrillary stains.
Type 2 muscle atrophy: disuse, cachexia, steroid-induced, myotonia congenita
Type 1 muscle may be preferentially atrophied in myotonic dystrophy type 1.
Stains
H&E stain: It is the most important stain used routinely and clearly shows the overall structure of the tissue in relation to the fibers, nuclei, fibrous and adipose tissue. It shows the presence of inflammatory cells, vacuoles, vascular and neural components. It can help in assessment of the size, shape, and cytoarchitecture of muscle fibers. Necrosis of muscle fibers, regeneration (basophilia), presence of internalized nuclei, dystrophic feature in supporting connective tissue and vasculature (vasculitis) is easily seen. Inflammatory infiltrates are easily appreciated with this stain. With the H&E stain, nuclei stain blue, the muscle fibers stain pink and the connective tissue stains a lighter pink. Basophilic fibers stain a light blue hue.
Gomori trichome stain: Similar to H&E, plus it is good to pick up mitochondria, (type 1 fibers) ragged red fibers associated with mitochondrial myopathies, tubular aggregates, nemaline rods, rimmed vacuoles, cytoplasmic bodies, myofibrillar myopathies, and IBM. It is sometimes easier to observe subtle increases in endomysial connective tissue with the modified Gomori trichrome technique in which the muscle fibers stain a greenish-blue color, and the collagen is a lighter but clearly distinguishable blue–green color. Nuclei stain red with the Gomori stain and the myelin of the nerve stains a foamy red color. Nerves may appear poorly stained in the absence of myelin. Abnormal accumulations of myofibrillar material may appear to be a darker green–blue color. A major application of the modified Gomori technique is the identification of red staining structures such as rods, cytoplasmic bodies, reducing bodies, abnormal mitochondria and the membranous myelin-like whorls of rimmed vacuoles. Mitochondrial accumulations appear as red aggregates of stain and the intermyofibrillar mitochondria appear as a series of fine dots throughout the fiber. Normal muscle fibers (especially aging) frequently show peripheral aggregates of mitochondria and care is needed not to over-interpret their significance.
Oxidative stains (NADH-TR, SDH, COX) are useful for identifying mitochondrial and intermyofibrillar network abnormalities.
NADH-TR: fiber type pattern, fibers with abnormal mitochondria, myofibrillar disruption, cores.
Type 1 muscle fiber is darkly stained
Type 2A muscle fiber is intermediately stained
Type 2B muscle fiber is lightly stained.
COX: fiber type pattern, fibers with abnormal mitochondria. It is encoded by mtDNA.
SDH: fiber type pattern; fibers with abnormal mitochondria. It is encoded by nuclear DNA.
COX + SDH: fibers devoid of cytochrome c activity appear blue; suggestive of mitochondrial myopathy with mtDNA mutations. Also aged muscle.
Myofibrillar adenosine-triphosphatase (ATPase) stains at various pHs are used to distinguish the different muscle fiber types.
The ATPase activity with myofibrillary stain for type I fibers is weak at alkaline pH (9.4), but strong at acidic pH (4.3); thus, the type 1 fibers appear pale at pH 9.4 and dark at pH 4.3, and the reverse is true of type 2 fibers. This distinction can be further refined by carrying out the ATPase reaction at pH 4.6, which permits differentiation of type IIA and type IIB fibers. A small percentage of fibers appear dark, strongly reactive at both acid and basic pH—and these are designated IIC fibers.
PAS (periodic acid-Shiff) stain for glycogen. Fibers with excess glycogen are heavily stained red or "hot-pink." Fibers with loss of glycogen appear white. It is worth bearing in mind, however, that not only glycogen but other polysaccharides, as well as neutral mucopolysaccharides, muco- and glycoproteins, glycolipids and some unsaturated lipids and phospholipids are stained with this reaction. The specificity of the PAS reaction for glycogen may be checked by using α- amylase (diastase) digestion and the use of celloidin helps to retain the glycogen prior to exposure to PAS. Although glycogen storage may be rare, the PAS technique is also useful in revealing damaged and some denervated fibers in several disorders as these may be devoid of glycogen and appear white.
Type 2 muscle fibers appear "hot-pink" and more intense.
Type 1 muscle fibers appear white or pale rosy colored.
Oil Red O: Intracellular lipids seen as red dots. Lipid (oil red O)
Type 1 muscle fibers have more red dots (lipids)
Type 1 muscle fibers have sparse red dots.
Sudan black: As for oil red O but a black end product (phospholipid also stain black)
Phosphorylase stain: Absent in type V glycogenosis (McArdle disease).
Type 2 muscle fibers appear dark.
Type 1 muscle fibers appear pale.
Phosphofructokinase: Absent in type VII glycogenosis; fiber type pattern. It may be useful to study if a glycogenosis is suspected, but only a result of total absence can be relied on. A deficiency is difficult to access histochemically and requires biochemical analysis.
Acid phosphatase: High in lysosomal storage (type II) disorders and vacuolar myopathies; high in necrotic fibers; highlights lipofuscin. Not apparent in normal muscle but present in perinuclear regions where focal deposits of lipofuscin is present in adults; not seen in children. It also highlights the presence of macrophages. Acid phosphatase activity is also abundant in vitamin E deficiency and Batten disease and the deposits are autofluorescent. The color of the autofluorescence can be used to distinguish the two types of deposit as in vitamin E deficiency they are orange–yellow but yellow in Batten disease.
Alkaline phosphatase: It is found primarily in cell membranes where active transport processes occur, such as the endothelium of arterioles and the arterial part of capillaries, and also in endoplasmic reticulum, Golgi apparatus and pinocytotic vesicles. The reaction is usually negative in muscle fibers but may be positive in focal necrotic fibers in various disease situations, and in some regenerating or non- innervated fibers. Its major use is in the assessment of inflammatory myopathies when perimysial areas may be intensely stained (blood vessels and perimysium)
Acetylcholinesterase: Neuromuscular junctions, myotendinous junctions, vacuoles in X- linked myopathy with excess autophagy vacuoles (XMEA), denervated/non-innervated fibers positive.
Non-specific esterase: Neuromuscular junctions, myotendinous junctions, phagocytic areas, small angulated denervated fibers positive. A two-fiber pattern may also sometimes be seen.
Myoadenylate deaminase (MAD) stain for MAD deficiency (muscle pain syndromes, myoglobinuria). Absent/deficient in exertional myalgia (significance uncertain); fiber type pattern; tubular aggregates very darkly stained. Interpretation of the significance of a deficiency is hampered, however, by the presence of a common polymorphism in the normal population which obliterates the enzyme. A secondary reduction in enzyme activity may also occur for unknown reasons.
Amyloid deposition can be detected by using Congo red, crystal violet, rhodamine or immunostaining.
Verhoeff-van-Gieson stain: highlight connective tissue red, elastin and myelin black.
Immunohistochemistry also performed on frozen section: MAC, complement, immunoglobulin (IgM, IgG, IgA), MHC-1, T & B cell markers
Dystrophin immunostain: DMD and BMD
Merosin immunostain: congenital muscular dystrophy
Sarcoglycan immunostain: sarcoglycanopathy (LGMD)
Dysferlin immunostain: dsyferilnopathy (LGMD)
Caveolin-3
Emerin
Desmin
Titin
alpha-actinin
Nebulin
myosin
actin
MCH1 stain and MXA1 stain seen in DM even before there is sign of atrophy.
Muscle Histology
Under light microscopy, appears polygonal with visible, but not excessive amount of connective tissue. There should be no or few inflammatory cells or necrotic muscle fibers. The distribution of the muscle fiber types is assessed with myofibrillary ATPase at 3 pH: 4.3, 4.6, and 9.4. Muscle tissue exposed to these different pHs result in muscle fiber displaying distinct staining qualities based upon their biochemical substrate mechanisms. The checkerboard appearance of different muscle fiber types becomes apparent at 30 weeks. In normal muscle there is <12% difference in the largest mean fiber diameters between all 3 muscle fiber types.
Muscle fibers are polygonal in shape. Mean fiber diameter is 16 um @ 1 year and increases by 2 um until age of 5 years, and then 3 um until age 9 years. By 10 years mean diameter ranges 39 - 42 um. Normal adult size is reached by 15 years with mean diameter range 50 - 73 um depending on the specific muscle seen.
True target: neurogenic disorders
Internalized nuclei vs central nuclei (dystrophic pattern)
In DM, MAC stain is deposited around small blood vessels. In dystrophies like FSHD and dysferlinopathies MAC are reported around the sarcolemma.
In muscular dystrophies muscle fibers are more rounded than polygonal, more contracted and darker. Fibers with big nuclei.
Loss of myosin heavy chains on EM seen in CIM.
Paradigm of chemical energy conversion into mechanical energy within muscle fibers: Components
Excitation and contraction in muscle membranes
Muscle contractile mechanism
Supporting structural elements that allow the muscle to withstand mechanical stresses
Dystrophin, sarcoglycans, alpha-laminin which link the contractile proteins with the extracellular supporting structures.
Energy system that support the above systems (1 to 3).
Myopathies are logically categorized base on what part of the system is involved.
Muscles to biopsy
Muscle to biopsy for proximal myopathies: Biceps brachii, deltoid, triceps brachii, quadriceps.
Muscles to biopsy for distal myopathies: Forearm extensor muscles, tibialis anterior , gastrocnemius
Pathological analysis of tibialis anterior and gastrocnemius may be confounded by neurogenic changes secondary to asymptomatic lumbosacral radiculopathies or concomitant peripheral neuropathy.
Cervical paraspinal muscles: Isolated drop head.
Peroneus brevis and superficial fibular nerve biopsy: suspected vasculitis.
Handling muscle biopsy tissue wrapped lightly in saline moistened gauze to prevent overwetting or overdrying and should be immediately sent to pathology lab for processing. Do not put biopsy specimen on saline or fixative.
Type 1 muscle predominance on biopsy: Inherited myopathies (centronuclear, reducing bodies, RYR1 mutation, central core, CACNA1S congenital myopathy, CACNA1S congenital myopathy which is allelic to HypoKPP1, malignant hyperthermia, TTPP1), demyelinating neuropathies, CIDP, ACTA1, SBMA, ACTN2, LGMD1A, MYBPC3, PTPLA/HACD1, SECISBP2, SCN4A, SEPN1, STAC3, ZAK.