Acquired Myasthenia Gravis in a German Shepherd Dog
Michael Mastromauro Clinical Advisor: Dr. Andrea Johnston Pre-Clinical Advisor: Dr. Stephen Barr Senior Seminar paper Cornell University College of Veterinary Medicine October 26th, 2011 Key Words: Acquired myasthenia gravis, megaesophagus, seronegative myasthenia gravis, autoimmune disease, mycophenolate mofetil, German Shepherd

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Abstract A five-year old male castrated German Shepherd dog, presented to the Cornell University Hospital for Animals (CUHA) Emergency Service on September 11th, 2011 for hindlimb paresis and aspiration pneumonia. On initial assessment, the patient exhibited tetraparesis that worsened with activity. Repeat thoracic radiographs showed persistence of previously diagnosed megaesophagus and aspiration pneumonia. The patient was hospitalized and started on IV fluids and antibiotics and transferred to the Internal Medicine Service the next day. A Tensilon test was performed which was positive and treatment was initiated with pyridostigmine, dexamethasone, and mycophenolate mofetil for presumptive acquire myasthenia gravis. Anti-acetylcholine receptor antibody titers were submitted but the patient was deemed seronegative. Over the next 3 days, the patient improved and was discharged with near complete resolution of both the megesophagus and aspiration pneumonia. Using the above case as an example, the pathophysiology of acquired myasthenia gravis with a focus on diagnostic and treatment options will be discussed.

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Case Summary
A five-year old, 30 kg male castrated German Shepherd dog presented to the Cornell University Hospital for Animals (CUHA) Emergency Service on September 11th, 2011 for a several day history of generalized paresis and aspiration pneumonia. On September 2nd, 2011, the dog had a two day history of lethargy and inappetance which would resolve on its own without treatment. Four days later, he began violently retching and regurgitating which prompted presentation to a local emergency clinic which treated him for presumptive lungworm and discharged him that night. The following day, he became increasingly lethargic and continued to have episodes of regurgitation. That evening, he was brought to another emergency clinic which took two-view thoracic radiographs that revealed megaesophagus and aspiration pneumonia. Treatment with metoclopramide and doxycycline was initiated and the patient was once again discharged into the care of his owners. However, he continued to decline over the next few days eventually developing a hindlimb paresis which progressed to a tetraparesis. On September 11th, 2011, he was referred to the CUHA Emergency Service for further evaluation.
On presentation, the dog was minimally ambulatory, tachycardic, and tachypneic with increased lung sounds bilaterally. Point-of-care blood work, consisting of quick assessment tests (QATS) and an iSTAT, and urinalysis were performed which revealed no abnormalities. Radiographs revealed persistence of both the megaesophagus and a cranioventral airspace pattern (aspiration pneumonia) in the right middle lung lobe. A problem list was generated which contained three major problems: generalized paresis, megaesophagus, and a cranioventral lung airspace pattern. Generalized paresis can be caused by intracranial (brain tumor, encephalitis, etc.), neuropathic (cervical spinal cord lesion, polyneuropathy, etc.), neuromuscular (myasthenia gravis, botulism, tick-born paralysis, etc.), and myopathic (polymyositis, hypothyroidism, etc.)

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causes. Megaesophagus, particularly in a German Shepherd, is most often caused by myasthenia gravis (MG), though other differentials include hypoadrenocorticism, dysautonomia, and any cause of generalized paresis. A cranioventral lung airspace pattern seen on thoracic radiographs in a dog with a recent history of regurgitation or vomiting is highly suggestive of aspiration pneumonia, though other differentials can include bronchopneumonia, neoplasia, and hemorrhage. Taking into account the patient’s signalment, clinical signs, and problem list the top differential diagnosis for the dog’s disease process was acquired myasthenia gravis with associated megaesophagus and secondary aspiration pneumonia. He was admitted to the hospital and placed on IV fluids at 1.5 times maintenance, ampicillin (Unasyn®, 22 mg/kg IV every 8 hours), and a urinary catheter was passed to empty his bladder.
The following morning, the patient was transferred to the CUHA Internal Medicine service, at which time his physical and neurologic examinations remained unchanged. Bloodwork was submitted for a complete blood count (CBC), chemistry panel, free T4 and thyroid-stimulating hormone (TSH) levels, cortisol level, and anti-acetylcholine receptor antibody titer. All of these tests were within the reference range with the exception of a “stress leukogram” observed on the CBC. A urinalysis was submitted which also revealed no abnormalities. In order to help reach the presumptive diagnosis of MG, an edrophonium chloride (Tensilon) response test was performed. This test involves IV administration of Tensilon, an ultra-short acting acetylcholinesterase inhibitor (AChEI). The patient was pre-medicated with 0.6 mg of atropine subcutaneously (SQ) and then given 1.5 mg of Tensilon IV. After no response was initially seen, an additional 1.5 mg of IV Tensilon was given at which time a strong positive response was observed. This positive result was based on observation of the dog becoming

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ambulatory and walking approximately 6 meters before once again tiring. Based on this finding, the dog was presumptively diagnosed with MG.
Treatment was initiated with pyridostigmine (1 mg/kg PO every 8 hours), mycophenolate mofetil (16.6 mg/kg PO every 12 hours), and dexamethasone (0.2 mg/kg every 24 hours, for 3 days). He was also continued on IV Unasyn for the next 24 hours until being transitioned to amoxicillin/clavulanic acid (Clavamox®, 375 mg PO every 12 hours) the following day. To help prevent regurgitation, the patient was fed canned food in small meatballs twice daily. After finishing the meal, he was kept in a sitting position with his head held elevated for 10-15 minutes to promote esophageal emptying. A day after beginning therapy, the dog began showing signs of side effects from the pyridostigmine (hypersalivation, hyperlacrimation, mild bradycardia, diarrhea), which prompted reduction of his overall dose. This was accomplished by halving his afternoon does to 0.5 mg/kg, but leaving the morning and evening doses at 1 mg/kg.
Over the next two days, the dog continued to improve showing increased stamina and ability to ambulate. On the day of his discharge, four days after presentation, the patient was able to walk for several minutes before tiring and needing short periods of rest. He had not had a single episode of regurgitation since while in our care. A repeat left lateral thoracic radiograph was performed which showed near complete resolution of both the megaesophagus and aspiration pneumonia. He was discharged on the above described doses of pyridostigmine, mycophenolate mofetil, and two more weeks of Clavamox .
Discussion: Acquired Myasthenia Gravis
Myasthenia gravis is a neuromuscular disorder caused by a deficiency in nicotinic acetylcholine receptors (AChR) on the neuromuscular endplate.1-4 There are two recognized forms: a rare congenital form and the much more common acquired form. The congenital form,

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which will not be discussed in detail here, is seen most often in young (< 4 months old) terrier breeds and results from improper synthesis of the AChR.1
Acquired myasthenia gravis is an autoimmune disorder caused by a T-cell dependent, Bcell mediated process in which the patient’s immune system begins making antibodies (mainly IgG) directed against the nicotinic AChR.1 The disease is seen mostly in larger breed dogs (Newfoundlands, Great Danes, German Shepherds, Labrador Retrievers, etc.), though it can occur in any breed. A genetic predisposition towards the disease has been proven in the Newfoundland and Great Dane.7,8 There appears to be a bimodal distribution of the disease as it occurs most commonly in dogs less than 5 years and greater than 8 years of age. Males and females appear to be equally represented.1-3 Pathophysiology
In the normal dog, neuromuscular transmission is initiated when ACh within vesicles travels down the axon of a lower motor neuron and is then released into the neuromuscular junction. When a sufficient number of AChRs on a muscle fiber are bound by ACh, contraction is initiated. In order to ensure the signal is transmitted, an excess amount of ACh is released from the pre-synaptic nerve cell. This signal is ended with the aid of an enzyme, acetylcholinesterase (AChE), which is present at the neuromuscular junction and hydrolyzes most of the released ACh.1,2
In the myasthenic patient, the immune system makes antibodies directed against components of the nicotinic AChR, most commonly the α subunit.1-4 Subsequent disruption of the receptor is thought to occur via three main mechanisms: 1) complement-mediated lysis of the muscular end plate, 2) cross-linking of adjacent receptors leading to their internalization and degradation, and 3) blockage of the ACh binding site on the receptor itself.1,3 What results is a

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decrease in the total number of available AChRs to perpetuate muscular contraction and subsequent muscular weakness. Clinical Signs and Disease Forms
The most common clinical presentation is a generalized paresis that worsens with exercise. Other clinical signs depend on which muscle groups are affected.1-5 Megaesophagus occurs in an estimated 90% of MG cases and results in the common finding of regurgitation.1 The high rate of megaesophagus in canine myasthenic patients is thought to be due to the higher percentage of skeletal muscle within the canine esophagus as compared to humans and our other domestic species.2,3 Development of aspiration pneumonia, a serious and life-threatening complication of this regurgitation, is the most common reason patients with MG are euthanized at presentation and is a negative prognostic indicator.1,2
MG can be divided into four categories: 1) focal, 2) generalized, 3) fulminating, and 4) neoplastic.1,5 The focal form, accounting for 37-43% of cases, is characterized by involvement of only one muscle group. In people, and some dogs, the ocular muscles may be the only muscles affected. Focal involvement of the pharyngeal or laryngeal musculature may present as dysphagia or dysphonia. Occasionally, the esophagus may be the only organ affected.1
The generalized form, as seen in our patient, is the most common clinical presentation, accounting for an estimated 57-64% of cases. Generalized MG is characterized by weakness of multiple muscle groups. Many generalized myasthenics will initially develop hindlimb paresis that progresses to tetraparesis, the reason for which is not completely understood. The weakness becomes worse as the animal exercises and they may become non-ambulatory. Concurrent megaesophagus is almost invariably found at presentation.1-5

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Fulminant MG(also referred to as a “myasthenic crisis”) is characterized by a severe, acute onset of generalized clinical signs. Aside from the signs associated with tetraparesis and megaesophagus, the respiratory muscles are also usually involved, leading to severe impairment of ventilation. Subsequently, these patients often present in respiratory distress potentially made worse by the presence of aspiration pneumonia. Fulminant myasthenics often have to be placed on mechanical ventilation.1,3,4 Prognosis for this form of MG is grave with many owners choosing to euthanize at presentation. Thankfully, fulminant MG accounts for less than 5% of documented cases.1
Some authors choose to place MG associated with neoplastic disease into yet a fourth category. In both human and canine myasthenics, the presence of a thymoma has been the most common neoplasia implicated.1,3 One causal theory is due to the thymus’s function as an important center for T cell maturity. Furthermore, it has been theorized that the neoplastic cells of epithelial origin that make up the tumor share similar epitopes to the AChR, leading to induction of antibodies against the receptors themselves.8 Other tumor types that have been found in association with MG are lymphoma and cholangiocellular carcinoma.1 Thus, before entering into a lengthy, expensive treatment interval, it is advised that advanced diagnostics and imaging be performed, particularly in older dogs who present for MG. Diagnosis
Once suspicion of MG has been raised by the clinical signs and initial diagnostics (bloodwork, radiographs, ultrasound), achieving a diagnosis should be pursued so that treatment may quickly be initiated.1,3, 9 In true myasthenics, bloodwork is often unremarkable unless concurrent disease is present.1 Neurologic examination may also be unrewarding, though occasionally the ability to cause tiring of the palpebral, menace, or patellar reflexes may be

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appreciated.3-5 The two most common tests used for MG diagnosis are the edrophonium chloride response (Tensilon) test and the anti-AChR antibody test.
As discussed above, edrophonium chloride is an ultra-short acting AChEI. It acts to prevent the hydrolysis of the excess ACh released from the presynaptic nerve terminal.1-5 This allows for there to be more available ACh at the neuromuscular junction to bind to the limited number of receptors of the muscle cell membrane and stimulate contraction. The dose of edrophonium that is commonly employed is 0.1-0.2 mg/kg IV.1 Often, half of this dose administered, and then the dog is assessed for improvement, usually by attempting to walk the patient in generalized cases. If no improvement is seen, then the other half is administered and assessment is repeated. Observation of improvement is interpreted as a positive result and is highly suggestive of MG. Unfortunately, the test is neither sensitive nor specific. False positives may occur in other neuromuscular diseases that result in paresis. False negatives are common, and may be due to a lack of remaining available AChR’s to elicit a response.1,2
Though the Tensilon test is useful for establishing a quick, presumptive diagnosis, definitive diagnosis is based on demonstrating a positive anti-AChR antibody titer.3-5 This test is both sensitive and specific and proves the presence of circulating antibodies against AChR.1 Diagnosis is based on a titer above 0.6 nmol/L. However, the magnitude of the rise in titer does not correlate with the severity of clinical disease.1-3 Though this test is the gold standard for MG diagnosis, an estimated 15% of myasthenic dogs may be seronegative, as was the case in our patient. Theories for why this may occur include samples taken too early in the course of the disease and that the AChR may not be the only protein targeted in MG. In human myasthenics, antibodies against other skeletal muscle proteins, including myosin, actin, titin, muscle specific

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receptor tyrosine kinase (MuSK), and the ryanodine receptor (RyR) have been described and may play a significant role in the overall course of the disease.1 Treatment
Various options are available for the treatment of MG. Most protocols include at the minimum some type of maintenance AChEI +/- an immunosuppressant(s). The therapeutic plan should be tailored to each individual patient and, particularly in the early stages, regular dose adjustments may be warranted. In most patients, rapid reduction in the degree of clinical signs is vital in order to prevent life-threatening complications. In contrast to human myasthenics, wellmanaged canine patients commonly will enter into clinical remission and may eventually be able to be weaned off all medications.1-5, 9-11 Acetylcholinesterase Inhibitors (AChEIs)
The two most commonly used AChEIs are pyridostigmine and neostigmine. They exert their therapeutic effects by competing with ACh as a substrate for AChE. As in the Tensilon test, this allows for a higher concentration of available ACh. Pyridostigmine is usually preferred as it is normally better tolerated with fewer side effects. Its recommended dose is 0.5-3.0 mg/kg PO every 8 to 12 hours.1 Though an effective treatment for MG, it is important to realize that AChEIs are merely a symptomatic treatment, doing nothing to combat the autoimmune origins of the disease. However, as discussed above, many canine myasthenics will eventually undergo spontaneous remission, making symptomatic treatment sufficient even when utilized alone. Side effects of AChEIs are common as a result of the effects of the excess ACh on the muscarinic AChRs of the parasympathetic nervous system and induction of the SLUDD pathway. Side effects include hypersalivation, hyperlacrimation, diarrhea, and bradycardia.1-5,9

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Immunosuppressants Being autoimmune in origin, treatment with immunosuppressants/immune modulators is
often undertaken in MG. As with many autoimmune disorders, corticosteroids, such as prednisone, at immunosuppressive doses (0.5-1.0 mg/kg every 24 hours) are often employed, particularly early on in the disease process to help gain rapid control over clinical signs.1 Though beneficial, corticosteroids also have a number of side effects that are displeasing to owners (hyperactivity, polyuria, polydipsia, etc.) when used long term. Thus, many other immunosuppressants have been utilized, either alone or as a means of lowering the dose of concurrently used corticosteroids. Commonly used drugs include azathioprine, cyclosporine, leflunomide, and mycophenolate mofetil (MMF).1,3,9 Based on recommendations from our neurologists, we chose MMF.
Mycophenolate works by inhibiting purine synthesis in both T and B lymphocytes via noncompetetive, reversible inhibition of inosine monophosphate dehydrogenase. This enzyme is necessary for the synthesis of guanosine triphosphate in what is termed the “de novo” pathway of purine synthesis.1,10,11 In most cells, a backup mechanism termed the “salvage” pathway exists. However, lymphocytes are only able to utilize the “de novo” pathway, a fact that is exploited in the use of MMF. Some reports have documented rapid, complete remission in only a few days with MMF, while others have showed no significant advantage to the use of the drug over AChEIs alone.10,11 Published doses range from 7-16 mg/kg PO every 12 hours and side effects are dose-dependent ranging from gastrointestinal upset to severe bone marrow suppression.1
Other Considerations Management of acquired megaesophagus is an important consideration, particularly in
cases where resolution does not occur. It is important to educate clients about the importance of limiting episodes of regurgitation to help prevent development of aspiration pneumonia. Often,

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patients will need to be fed multiple small meals throughout the day and be positioned in an elevated sitting position or completely vertical so that the esophagus is perpendicular to the floor, in order to promote esophageal emptying.2,3 This may be quite challenging for even the most dedicated owners, though apparatuses such as the “Bailey chair” are available to assist in this endeavor. Development of aspiration pneumonia is a serious and life-threatening complication of repetitive regurgitation and needs to be addressed immediately. In fact, aspiration pneumonia is the most common factor leading to euthanasia in the myasthenic patient.2 Therefore, early recognition and treatment of aspiration pneumonia is vital for long-term prognosis.
Another complication is the development of drug-related side effects, particularly those stemming from inadvertent overdosing of AChEIs. Overwhelming stimulation of both nicotinic and muscarinic acetylcholine receptors can lead to severe clinical signs and a presentation referred to as a “cholinergic crisis”. These patients present with similar clinical signs to those in a “myasthenic crisis” including generalized paresis and respiratory distress.1-3,9 This occurs because chronic overstimulation of AChRs will eventually lead to them becoming refractory to the effects of ACh binding.2 Distinguishing between cholinergic and myasthenic crisises in these patients is extremely important so that swift initiation of treatment can be undertaken. The most common method of diagnosis is by performing the Tensilon test. Worsening of clinical signs is indicative of a cholinergic crisis, while improvement would be supportive of a myasthenic crisis. In cases of cholinergic crisis, atropine is normally the drug of choice for quick resolution of clinical signs.1
Prognosis Prognosis for most myasthenic patients is quite guarded in the early stages, as
approximately 50% of patients may be euthanized at presentation depending on the severity of

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their clinical signs.2,3 For those patients whose owners choose to initiate treatment and survive past the first 2 weeks, prognosis is upgraded to fair. Obviously, this is highly dependent on owner compliance and successful adjustments of drug dosages in the early stages. It is important to remember that in canine myasthenics, spontaneous remission is possible. Many patients may eventually be able to be weaned off of their medications, eliminating undesirable side effects as well as financial burden. In one study, the time required to achieve spontaneous remission was estimated to be on average 6.4 months.1,2 The most common method of tracking response to treatment is by serial anti-AChR antibody titers, a practice that poses is challenging in seronegative myasthenics.1-5, 9
Conclusion Myasthenia gravis is a disease of severe neuromuscular weakness that may be easy to
diagnose but challenging to manage. Successful treatment is often the result of dedicated cooperation between clients and their veterinarians. Even with initiation of treatment, over 50% of myasthenic patients will be euthanized or die due to complications within a year of diagnosis.2,3 As more and more research is being conducted on both human and canine myasthenic disease forms, improved recommendations will be available for long-term treatment. The ability of the canine myasthenic patient to achieve spontaneous remission is an important factor that differentiates them from their human counterparts. Though many treatment options are available, particularly in regards to immunosuppressive agents, there is not a consensus on the best treatment combination to pursue. Research is currently underway to evaluate the efficacy of new treatment modalities including the possibility of one day administering “vaccines” which will block the ability of immunoglobulins to bind to their targets with the hope of diminishing clinical signs while experiencing fewer side effects.

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References
1. Khorzad, R, Whelan, M, Sisson, A, et al. Myasthenia gravis in dogs with an emphasis on treatment and critical care management. J Vet Emerg and Crit Care 2011; 21(3):193-208. doi: 10.1111/j.14764431.2011.00636.x. 2. De Lahunta, A, Glass, E. Lower Motor Neuron: Spinal Nerve, General Somatic Efferent System,. In: Veterinary Neuroanatomy and Clinical Neurology. 3rd ed. St. Louis: Elsevier 2009; 93-95. 3. Thomas WB. Myasthenia Gravis. In: Cote E. Clinical Veterinary Advisor: Dogs and Cats. 2nd ed. St. Louis: Elsevier 2011; 736-737. 4. Dewey, C.W. Acquired Myasthenia Gravis in Dogs -Part I. The Compendium 1997; 19 (12): 1340-1353. 5. Dewey CW, Bailey CS, Shelton GD, et al. Clinical Forms of Acquired Myasthenia Gravis in Dogs: 25 Cases (1988-1995). J Vet Intern Med. 1997; 11:50-57. 6. Lipsitz D, Berry JL, Shelton GD. Inherited predisposition to myasthenia gravis in Newfoundlands. J Am Vet Med Assoc 1999; 215: 946-958. 7. Kent M, Glass EN, Acierno M, et al. Adult onset acquired myasthenia gravis in three Great Dane littermates. J Small Anim Pract 2008; 49:647–650. 8. Romi F. Thymoma in myasthenia gravis: from diagnosis to treatment. Autoimmune Diseases 2011; 2011: 1-5. doi:10.4061/2011/474512. 9. Dewey, C.W. Acquired Myasthenia Gravis in Dogs -Part II. The Compendium 1998; 20 (1): 47-57. 10. Dewey CW, Boothe BM, Rinn KL, et al. Treatment of a Myasthenic Dog with Mycophenolate Mofetil. Vet Emerg Crit Care. 2000; 10: 177-187. 11. Dewey CW, Cerda-Gonzalez S, Fletcher DJ, et al. Mycophenolate mofetil treatment in dogs with serologically diagnosed myasthenia gravis: 27 cases (1999-2008). J Am Vet Med Assoc 2009; 236 (6): 664-668.